i
Acknowledgements
Despite being of few words, I have been lucky enough to be surrounded by great people, that have helped me overcome any hurdle encountered, whether in my scientific career or in life.
I would like to start by thanking my supervisors, Dr. Fábio Fernandes and Professor Ana Coutinho, for giving me a chance, taking me in and guiding me through this journey. I would like to extend a special thank you to Fábio, who gave me all the tools and knowledge to be successful. I hope I have met most of your expectations. I would also like to thank Professor Manuel Prieto, for welcoming me into his group (Biological Fluorescence group at CQFM) and showing his support along this year. I would like to thank Dr. Arsénio Fialho, Dr. Nuno Bernardes and the Institute for Bioengineering and Biosciences, for allowing us to use their facilities and offering their help for our protein expressions and purifications.
To Fundação para a Ciência e Tecnologia (FCT) I would like to acknowledge and thank the financial support on the project RECI/CTM-POL/0342/2012.
To every current and former member of CQFM that I encountered, I express my utmost gratitude. I was quickly integrated into, what felt like, a second family, something I had never before experienced. To my officemates (and honorary office members), I am grateful for our amazing discussions, our adventures, our victories and our failures. I am proud to call you all my friends.
To Tânia Sousa, I could not be more grateful for your support. Not only did you offer unconditional help and outstanding mentorship, you were a great friend. Safe to say, everything would have been immensely more difficult without your presence.
To those who I call my family, I would like to offer some special words and gratitude:
To my closest friends, who to me are like brothers and sisters, and have backed me up since we were boys, I am forever indebted. Only those who were there to see how low we have been can truly appreciate how far we have gotten.
To my partner in crime, who was always there when I needed her most, for all the love and support, for the help and cheering up and for the occasional patching of a sinking ship, during what was a rough but incredible year.
To my parents, who always did their best to help me succeed. For putting up with all my odds and quirks and still offering unconditional love. I only am who I am because of you.
To my grandmother, who despite coming from a completely different world, with different truths and beliefs, always offered me pure love.
iii
Resumo
PI(4,5)P2 é um importante fosfoinositol, localizado no folheto interno das células eucariotas, que
possui um grupo inositol hidrófilo, carregado negativamente. As características biofísicas particulares do PI(4,5)P2 permitem que este consiga recrutar, regular e interagir com várias proteínas sinalizadoras.
Este fosfolípido tem um papel central na regulação da dinâmica da membrana plasmática, em eventos como a exocitose e a endocitose, participa como substrato na produção de segundos mensageiros em vias de sinalização, como a catalisada pelo fosfolipase C, sendo ainda um componente crucial na promoção do recrutamento de proteínas para o folheto interno da membrana plasmática.
A interação de catiões divalentes, especialmente o cálcio, com PI(4,5)P2, tem sido o foco de
vários estudos. O cálcio é um catião divalente, com uma concentração intracelular baixa e altamente regulada. Este catião desempenha um papel crucial como elemento nas cascatas de transdução de sinal e como cofator importante para o funcionamento de várias proteínas. O grupo inositol fosforilado do PI(4,5)P2 participa em fortes interações electroestáticas com catiões divalentes, induzindo a formação
de micro domínios (clusters) de PI(4,5)P2 e influenciando a sua distribuição de carga. Este efeito é
observado mesmo em concentrações fisiológicas de PI(4,5)P2 e cálcio. No entanto, o impacto dos catiões
divalentes na interação de PI(4,5)P2 com as proteínas às quais este se liga e através das quais este
participa em várias vias de sinalização, só recentemente tem sido alvo de estudo.
De modo a aprofundar os conhecimentos nesta área, neste trabalho estudámos dois modelos proteicos com a capacidade de se ligarem a PI(4,5)P2 através de diferentes mecanismos, PH-YFP e
PBP-10. PH-YFP é uma proteína de fusão que consiste num domínio YFP, com a capacidade de dimerizar, ligado a um domínio PLCδ1-PH. O domínio PLCδ1 PH liga-se ao PI(4,5)P2 com elevada afinidade e
especificidade, através do reconhecimento estereoespecífico do grupo hidrófilo do PI(4,5)P2. O
PBP-10, por sua vez, é um péptido policatiónico que interage com o PI(4,5)P2 através de interações
electroestáticas não especificas. Utilizando uma combinação de métodos de espectroscopia e de microscopia confocal de fluorescência, utilizámos estes dois modelos proteicos de modo a determinar a influência de concentrações fisiológicas de cálcio nas interações PI(4,5)P2– proteína e na organização
membranar das proteínas ligantes de PI(4,5)P2.
Os estudos iniciais realizados com PH-YFP, confirmaram, através de ensaios de polarização de fluorescência e de histograma de contagem de fotões (PCH), que o PH-YFP dimeriza na membrana. No entanto, na presença de 100 μM Ca2+, esta dimerização é inibida reversivelmente. Este efeito não se
deve a uma menor concentração da proteína associada às membranas pois é observado mesmo na presença de concentrações saturantes de lípido. Por outro lado, os resultados obtidos por espectroscopia de correlação de fluorescência (FCS) mostram que o PH-YFP difunde na membrana de forma significativamente mais lenta na presença de cálcio, confirmando que esta proteína se liga e permanece associada aos clusters de PI(4,5)P2 formados a estas concentrações. A inibição da dimerização de
PH-YFP na membrana, na presença de cálcio, pode ser explicada através de um modelo em que os clusters de PI(4,5)P2 são suficientemente pequenos para tornar improvável a associação de mais que um
PH-YFP em simultâneo no mesmo cluster, efetivamente diminuindo a probabilidade de ocorrência de interações proteína-proteína na membrana. Neste modelo, a agregação de PI(4,5)P2 por cálcio, promove
um “sequestro” de proteínas com afinidade para este fosfolípido, diminuindo a disponibilidade destas para estabelecer interações com outros componentes membranares.
Utilizando a técnica de PCH, foi possível quantificar ainda a oligomerização de PH-YFP, confirmando-se que esta é dependente da concentração de cálcio. A 20μM de Ca2+,a fração de proteína
iv
oligomerizada, que fora detetada na ausência de cálcio, é praticamente nula. No entanto, a 50μM Ca2+ e
100μM Ca2+ é detetada uma ligeira recuperação desta população. Esta observação parece sugerir que o
tamanho dos clusters de PI(4,5)P2 aumentam com a concentração de cálcio, permitindo a incorporação
de várias proteínas no mesmo domínio e consequente oligomerização. No entanto, é necessário realizar estudos adicionais de modo a caracterizar este mecanismo mais detalhadamente. O conjunto destes resultados sugere que, in vivo, este mecanismo poderá afetar uma variedade de proteínas, podendo atuar como um “interruptor”, inibindo ou promovendo a interação entre proteínas.
Usando microscopia confocal de fluorescência, determinámos ainda que a afinidade de PH-YFP para com GUVs contendo PI(4,5)P2, varia também com a concentração de cálcio. Foi observada uma
resposta bifásica em que até 100μM Ca2+ se deteta um aumento da afinidade de PH-YFP. No entanto,
para concentrações mais elevadas de cálcio, a afinidade diminui para níveis ligeiramente mais baixos do que na ausência do catião. A maior afinidade da proteína para membranas a baixas concentrações de Ca2+, coincide com a formação de clusters de PI(4,5)P
2 e por isso poderá ser resultado de uma alteração
de conformação ou exposição do grupo hidrófilo do PI(4,5)P2 quando incluído nestes agregados. Por
outro lado, a diminuição da afinidade do PH-YFP para membranas a concentrações elevadas de PI(4,5)P2,estará muito provavelmente associada a um efeito de blindagem de carga ao PI(4,5)P2 pelo
cálcio, que interfere nas interações electroestáticas com o PH-YFP.
Nos estudos realizados com o péptido PBP-10, analisou-se o efeito do cálcio na partição do péptido para as membranas lipídicas, usando métodos de espectroscopia de fluorescência. Na presença de 100 μM Ca2+, observámos uma redução para metade no coeficiente de partição do péptido para LUVs
contendo 5% PI(4,5)P2. Esta redução não foi observada quando se testaram LUVs contendo 20% PS,
mantendo-se a densidade de carga média da superfície da membrana. Estes resultados sugerem que a partição do PBP-10 estará a ser afetado pelo efeito de blindagem de carga pelo cálcio ao PI(4,5)P2, tal
como observado para PH-YFP a concentrações superiores a 100 μM Ca2+. Uma vez que na interação
entre PBP-10 e PI(4,5)P2, as contribuições electrostáticas são dominantes para definir a afinidade entre
as moléculas, o efeito inibidor do cálcio ocorre a concentrações mais baixas.
Estudos subsequentes de FRET sobre a interação do PBP-10 com derivados fluorescentes dos dois componentes individuais dos modelos de membrana usados nestes estudos (TF-PI(4,5)P2 e
Bodipy-PC), na presença e ausência de cálcio, confirmaram a diminuição da interação do péptido com o PI(4,5)P2. Estes dados estão de acordo com a hipótese da blindagem de carga pelo cálcio ao PI(4,5)P2.
Experiências controlo de FRET confirmaram que não houve uma diminuição significativa da associação do péptido à membrana, levando a concluir que o cálcio leva a uma diminuição do PI(4,5)P2 sequestrado
pelo PBP-10.
Neste trabalho iniciou-se ainda o estudo de outra proteína ligante de PI(4,5)P2, o HIV-1 Gag,
com um mecanismo de interação membranar bastante mais complexo que o dos domínios PLCδ1 PH ou do péptido PBP-10. Ao contrário destes modelos proteícos, o Gag apresenta uma interação tripartida com a membrana, sugerindo que as interações PI(4,5)P2 – proteína e a organização membranar da
proteína serão dependentes também da composição membranar e dos tipos de cadeias acilo do PI(4,5)P2.
Neste contexto, iniciámos um processo de otimização da expressão, purificação e marcação com sonda fluorescente da proteína Gag para utilização neste projeto.
v Em suma, no conjunto dos estudos que constituem esta dissertação, foi observado que:
i. PH-YFP (modelo de ligação ao PI(4,5)P2 de alta afinidade) interage fortemente com os
clusters de PI(4,5)P2, a concentrações fisiológicas de cálcio e PI(4,5)P2, e não consegue
sequestrar o lípido destes domínios;
ii. As variações dos níveis de cálcio dentro da sua gama de concentrações intracelulares, aparentam não só influenciar a compartimentalização de PH-YFP nos clusters de PI(4,5)P2, como também de influenciar as interações proteína-proteína;
iii. O cálcio induz um efeito de blindagem da carga negativa do grupo hidrófilo do PI(4,5)P2, influenciando a afinidade e a ligação à membrana das suas proteínas ligantes.
iv. Diferentes mecanismos de interação dos modelos proteicos com o PI(4,5)P2 revelam
diferenças cruciais relativamente à sua sensibilidade ao cálcio.
Os resultados apresentados demonstram que a regulação indireta das interações proteína-PI(4,5)P2 pelo Ca2+ é mais complexa do que inicialmente considerado. Os dois mecanismos descritos,
através dos quais esta regulação poderá ocorrer, deverão atuar concertadamente, criando um nível adicional de regulação das proteínas ligantes de PI(4,5)P2,através do qual as flutuações dos níveis
intracelulares de cálcio são associadas a variações da afinidade e da organização destas proteínas pelo PI(4,5)P2.
É de prever que estes efeitos deverão ser especialmente importantes na vizinhança de canais de cálcio, onde as flutuações temporárias de concentração de cálcio são muito mais elevadas. É também de frisar que estes efeitos, afetarão diferentes proteínas de diferentes modos, podendo levar a uma complexa regulação de vias de sinalização. Com isto em mente, o nosso objetivo seguinte será caracterizar também o efeito do catião Mg2+, que possuindo muito menor afinidade para PI(4,5)P2 que o Ca2+, é também
capaz de induzir a segregação lateral destes fosfolípidos, ainda que a concentrações iónicas bastante superiores.
Palavras-chave:
PI(4,5)P2
Cálcio
Domínios Lipídicos
Espectroscopia e microscopia de fluorescência Interações Proteína-Lípido
vii
Abstract
The interaction of divalent cations with PI(4,5)P2, especially calcium, has been the focus of
intensive research in recent years. The negatively charged headgroups of PI(4,5)P2 are able to establish
strong electrostatic interactions with divalent cations, inducing the formation of PI(4,5)P2 nanodomains
even at physiological concentrations of PI(4,5)P2 and calcium. However, the direct impact of divalent
cations on the interaction of PI(4,5)P2 with binding proteins has received little attention up until recently.
Here, we studied two different proteins that have the ability to bind to PI(4,5)P2 specifically and
non-specifically, namely, PH-YFP and PBP-10 respectively. A combination of complementary fluorescence spectroscopy and microscopy techniques were used to determine the influence of calcium on PI(4,5)P2
-protein interactions and membrane organization of PI(4,5)P2 binding proteins. Our results show that, in
model membranes, PI(4,5)P2 binding proteins interact with calcium-induced PI(4,5)P2 clusters and are
not able to fully sequester the bound phospholipid from these structures. This stable interaction was shown to influence not only protein membrane diffusion but also protein – protein interactions after membrane binding, suggesting that this mechanism could act as an important regulatory mechanism. It was also found that calcium can regulate PI(4,5)P2 -protein affinity. The specific PLCδ1 PH
protein-PI(4,5)P2 interaction shows a complex biphasic response to calcium, with higher membrane binding
affinity at low concentrations of the divalent cation, and lower affinity at higher concentrations. While the observation that calcium can act as an inhibitor of protein-PI(4,5)P2 interactions due to effective
charge shielding of the phosphorylated inositol ring, is not unexpected, the complex response shows an opposite effect at low calcium levels. This is likely due to a change in the PI(4,5)P2 headgroup
conformation or exposure within the clusters, which increases affinity of the protein for PI(4,5)P2. We
propose that at lower Ca2+ levels, the impact of PI(4,5)P2 shielding within the clusters is greatly
compensated by a change in headgroup orientation, while at higher concentrations of the cation, the latter mechanism dominates. For PBP-10, since interactions are not dependent on binding site recognition, the electrostatic interactions dominate and its membrane binding affinity is solely influenced by PI(4,5)P2 shielding. After studying these two proteins, we aimed to extend our studies on the influence of calcium on PI(4,5)P2-protein interactions to a more complex protein system, HIV-1
Gag. To achieve this, we started the optimization of Gag expression, purification and fluorescent labelling. Altogether, the results presented here suggest a new regulatory mechanism, through which fluctuations in calcium concentration can impact protein-plasma membrane affinity and organization of PI(4,5)P2 binding proteins.
Keywords:
PI(4,5)P2
Calcium Lipid Domains
Fluorescence spectroscopy and microscopy Lipid – protein interactions
ix Table of Contents Acknowledgements ...i Resumo ... iii Abstract ... vii
Table of Contents ...ix
List of Figures ... xii
List of Tables ... xiv
List of Symbols... xv
List of Abbreviations ... xvii
1. Introduction ... 1 1.1. Biomembranes ... 3 1.1.1. Biomembrane Structure ... 3 1.2. Phosphoinositides ... 4 1.3. PI(4,5)P2 ... 5 1.3.1. PI(4,5)P2 metabolism ... 6
1.3.2. PI(4,5)P2 lateral organization ... 6
1.3.3. PI(4,5)P2 sequestration by proteins ... 7
1.3.4. PI(4,5)P2 and divalent cations ... 7
1.3.5. PI(4,5)P2 and protein interactions ... 9
1.3.5.1. Pleckstrin homology (PH) domains... 10
1.3.5.2. Polyphosphoinositide-binding peptide, PBP-10 ... 11
1.3.5.3. Influence of Ca2+ in PI(4,5)P 2-protein and protein-protein interactions ... 12
1.4. Human Immunodeficiency virus type 1 (HIV-1) ... 12
1.4.1. HIV-1 replication cycle ... 12
1.4.2. Gag precursor protein ... 13
1.4.2.1. Gag membrane targeting and binding ... 14
1.4.2.2. Gag interaction with PI(4,5)P2 ... 15
1.4.2.3. Role of lipid domains in the interaction of Gag with the plasma membrane 16 1.4.2.4. Gag multimerization and assembly ... 17
1.5 Objectives ... 18
2.Materials and Methods ... 19
2.1. Chemicals ... 21
2.1.1. Reagents... 21
2.1.2. Fluorescent Probes ... 21
x
2.2. Biological materials ... 22
2.2.1. Expression and purification of PH-YFP ... 22
2.2.2. Expression and purification of HIV-1 Gag ... 23
2.3. Model membrane systems... 23
2.3.1. Large unilamellar vesicles (LUVs) ... 23
2.3.2. Giant unilamellar vesicles (GUVs) ... 23
2.3.3. Lipid quantification ... 24
2.4. Calcium quantification ... 25
2.5. Absorption and fluorescence measurements ... 25
2.5.1. UV visible spectroscopy ... 25
2.5.2. Steady-state fluorescence spectroscopy ... 25
2.5.2.1. Steady-state fluorescence anisotropy ... 26
2.5.2.2. Förster resonance energy transfer (FRET) ... 27
2.5.2.2.1. homoFRET ... 29
2.5.3. Confocal fluorescence microscopy ... 29
2.5.3.1. Fluorescence Fluctuation Spectroscopy (FFS) ... 29
2.5.3.1.1. Fluorescence Correlation Spectroscopy (FCS) ... 30
2.5.3.1.2. Photon Counting Histogram (PCH) ... 32
2.6. Experimental procedures ... 35
2.6.1. Evaluation of membrane-associated PH-YFP oligomerization in the presence and absence of Ca2+ ... 35
2.6.1.1. Steady-state fluorescence studies of PH-YFP membrane partition and aggregation in the presence and absence of Ca2+... 35
2.6.1.2. Fluorescence fluctuation spectroscopy studies of PH-YFP aggregation in GUVs in the presence and absence of Ca2+ ... 36
2.6.1.3. Fluorescence confocal imaging and quantification of PH-YFP membrane binding in GUVs ... 36
2.6.2. Steady-state fluorescence studies of PBP-10 membrane partition in the presence and absence of Ca2+ ... 36
2.6.2.1. Evaluation of PBP-10 membrane partition through steady-state fluorescence intensity measurements. ... 37
2.6.2.2. FRET studies on PBP-10 interaction with PI(4,5)P2 containing LUVs. ... 37
2.6.3. Characterization of the purified Gag protein. ... 38
2.6.3.1. Evaluation of Gag interaction with PI(4,5)P2 containing LUVs. ... 38
2.6.3.2. Characterization of the purified Gag protein through FCS. ... 38
3. Results ... 39
xi 3.1.1. PH-YFP partitions to PI(4,5)P2 containing membranes and undergoes homoFRET in
the absence of calcium. ... 41
3.1.2. Fluorescence confocal microscopy and FFS studies of the effect of PI(4,5)P2 clustering on PH-YFP oligomerization. ... 46
3.1.2.1. PI(4,5)P2 clustering affects PH-YFP membrane diffusion ... 46
3.1.2.2. Fraction of oligomerized PH-YFP is higher in the absence of calcium. ... 48
3.1.2.3. Calcium influences PH-YFP affinity towards PI(4,5)P2 containing GUVs. .... 50
3.2. Effect of calcium on PBP-10 interactions with PI(4,5)P2 containing membranes ... 53
3.2.1. Effect of calcium on PBP-10 partition to negatively charged membranes. ... 53
3.2.2. FRET studies of the influence of calcium on PBP-10 interaction with the different membrane components ... 56
3.3. Optimization of the expression and purification of HIV-1 Gag precursor protein as a step towards the study of the influence of PI(4,5)P2 modulators on Gag oligomerization. ... 58
3.3.1. Initial optimizations of the expression and purification of HIV-1 Gag ... 58
3.3.2. Evaluation of Gag and it’s affinity towards PI(4,5)P2 containing membranes through fluorescence polarization studies... 62
3.3.3. Initial characterization of the purified Gag protein through FFS ... 64
4. Discussion ... 65
xii
List of Figures
Figure 1.1 An updated view of the fluid-mosaic model. ... 4
Figure 1.2 PI molecular structures. ... 5
Figure 1.3 Examples of PI(4,5)P2 roles at the plasma membrane. ... 6
Figure 1.4 PH domain 3D protein structure examples. ... 10
Figure 1.5 PBP-10 2D molecular structure. ... 11
Figure 1.6 HIV-1 replication cycle. The main steps are illustrated, as well as the major antiretroviral drugs next to the step of the cycle that they block.. ... 13
Figure 1.7 HIV-1 Gag protein structure and associated function. ... 14
Figure 1.8 Membrane binding model of MA:PS:PI(4,5)P2. ... 15
Figure 1.9 Model of membrane-bound HIV-1 Myr-MACA proteins viewed from the membrane side. ... 18
Figure 2.1 Schematic of the mixed coating used for immobilization. ... 24
Figure 2.2 Jablonski diagram showcasing a FRET pair.. ... 27
Figure 2.3 Fluorescence fluctuation spectroscopy (FFS). ... 30
Figure 3.1 PH-YFP partition and oligomerization anisotropy results. ... 43
Figure 3.2 PLCδ1 PH domain bound to Ins(1,4,5)P3. (PDB Entry: 1MAI). ... 45
Figure 3.3 Results from the FCS studies of PH-YFP. ... 47
Figure 3.4 Results from the PCH studies of PH-YFP. ... 50
Figure 3.5 Images taken from the equatorial plane of POPC:PI(4,5)P2 (95:5 molar ratio) GUVs in the presence of 250nM PH-YFP and varying concentrations of calcium. ... 52
Figure 3.6 Mean PH-YFP membrane fluorescence intensity in the absence and presence of several calcium concentrations.. ... 52
Figure 3.7 Results of PBP-10 Partition to membranes. (A) Normalized PBP-10 absorption (black) and fluorescence emission (red) spectra. ... 55
Figure 3.8 FRET study results on the influence of calcium on PBP-10 interaction with membranes... 57
Figure 3.9 Schematic of the main steps in the recombinant HIV-1 Gag expression and purification. ... 60
xiii Figure 3.10 Elution profiles from the Gag purification. ... 61 Figure 3.11 Fluorescence polarization characterization of the recombinant Gag proteins. ... 63 Figure 3.12 FFS Characterization of Gag and GagMBPHis in solution. ... 65 Figure 4.1 Schematic summary of how increasing calcium concentrations influence PI(4,5)P2
xiv
List of Tables
Table 1.1 Representative PI(4,5)P2 interacting domains and proteins. ... 9 Table 2.1 Probe spectral properties.. ... 21 Table 2.2 Fluorescence excitation and emission parameters used for the PH-YFP steady-state fluorescence experiments. ... 35 Table 3.1 Purification table summarizing the crucial steps in the Gag purification. ... 60
xv
List of Symbols
D Diffusion coefficient
E FRET efficiency
G Calibration factor
G(τ) Autocorrelation of fluorescence fluctuations I Fluorescence intensity
I0 Intensity value at the centre of the point spread function
J Overlap integral
k Orientation factor / number of photon counts kB Boltzmann’s constant
kd Dissociation rate constant
ket Energy transfer rate constant
ki Rate constant of donor excitation decay pathways
Kp Partition coefficient
n Refraction index
N Number of fluorescent particles
NA Avogadro’s number
pg PCH integration function using a 3D Gaussian approximation
Q PCH volume ratio
r Distance between donor and acceptor molecules <r> Steady-state fluorescence anisotropy
r0 Limiting anisotropy
R0 Foster Radius
Rh Hydrodynamic radius
S Axial to lateral dimension ratio
T Sampling time
V0 Observation volume
Vh Protein hydration volume
W(r) Observation volume profile w0 Lateral distance
z0 Axial distance
β PCH instrument and molecular factor
γ Incomplete Gamma function
γL Lipid molar volume
ε Brightness
η Solution viscosity
ηw Detection efficiency
θ Rotational correlation time
λ Wavelength
xvi
τD Translational diffusion time
φ Quantum yield
xvii
List of Abbreviations
(18:1) PI(4,5)P2 1,2-dioleoyl-sn-glycero-3-phospho-(1'-myo-inositol-4',5'-bisphosphate)
5ptaseIV Type IV inositol polyphosphate 5-phosphatase
A Acceptor
AFM Atomic force microscopy
AIDS Acquired immune deficiency syndrome
ANTH AP180 amino-terminal homology domain
APP Amyloid precursor protein
BSA Bovine serum albumin
Bodipy-PC
2-(4,4-difluoro-5-Methyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Dodecanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine
TopFluor-PI(4,5)P2 1-oleoyl-2-{6-[4-(dipyrrometheneboron difluoride)butanoyl]amino}hexanoyl-sn-glycero-3-phosphoinositol-4,5-bisphosphate
C1 Protein kinase C conserved region 1 domain
C2 Protein kinase C conserved region 2 domain
CA Gag capsid protein
CAM Calmodulin
CAP23 brain abundant membrane attached signal protein 1
CPSM Counts per second per molecule
CTD C-terminal domain
D Donor
DMF Dimethylformamide
ENTH Epsin amino-terminal homology domain
ESCRT Endosomal sorting complexes required for transport
EtOH Ethanol
FCS Fluorescence correlation spectroscopy
FERM Band 4.1, ezrin, radixin, moesin domain
FFS Fluorescence fluctuation spectroscopy
FRAP Fluorescence recovery after photobleaching
FRET Förster resonance energy transfer
Gag Group-specific antigen protein
GagMBPHis Gag-His tagged maltose-binding fusion protein
GagPol Group-specific antigen polyprotein
GAP43 Growth associated protein of 43kDa
GUV Giant unilamellar vesicle
HBR Highly basic region
HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
HIV-1 Type 1 human immunodeficiency virus
HOMO Highest occupied molecular orbit
Ins(1,4,5)P3 Inositol 1,4,5-trisphosphate
IPTG Isopropyl β-D-1-thiogalactopyranoside
LB Lysogeny broth
LUMO Lowest unoccupied molecular orbit
LUV Large unilamellar vesicle
xviii
MARCKS Myristoylated alanine-rich C kinase substrate
MD Molecular dynamics
MeOH Methanol
MHR Major homology region
MBPHis Histidine tagged Maltose-binding protein
NC Gag nucleocapsid protein
NMR Nuclear magnetic resonance
NTD N-terminal domain
OCRL Lowe oculocerebrorenal syndrome protein
PBP-10 Polyphosphoinositide-binding peptide
PBS Phosphate buffer solution
PC Phosphatidylcholine
PE Phosphatidylethanolamine
PH Pleckstrin homology domain
PH-YFP Phospholipase C δ1 subtype – yellow fluorescent fusion protein
PI Phosphoinositide
PI(3,4,5)P3 Phosphatidylinositol (3,4,5)-trisphosphate
PI(4)P Phosphatidylinositol (4)-phosphate
PI(4,5)P2 Phosphatidylinositol (4,5)-bisphosphate
PI3K Phosphatidylinositol (4,5)-bisphosphate 3-kinase
PIP4K Phosphatidylinositol 5-phosphate 4-kinase
PIP5K Phosphatidylinositol 4-phosphate 5-kinase
PKC Protein kinase C PLC Phospholipase C PLCδ Phospholipase C δ subtype POPC 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine POPS 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine PS Phosphatidylserine
PSF Point spread function
PTEN Phosphatase and tensin homologue on chromosome 10
PVA Polyvinyl alcohol
PX Phox domain
PtdIns Phosphatidylinositol
SB Super broth
SD Standard deviation
SEM Standard error of the mean
SNARE Soluble NSF attachment protein receptor
SP1 Gag spacer peptide 1
SP2 Gag spacer peptide 2
STED Stimulated emission depletion
STORM Stochastic optical reconstruction microscopy
TEV Tobacco etch virus
TRIS tris(hydroxymethyl)aminomethane
VLP Virus like particle
3
1.1. Biomembranes
Biological membranes are complex structures tasked with enclosing the cell and separating it from the surrounding environment. In eukaryotic cells, they also delimit spatially localized compartments that segregate processes and components for the purposes of increased efficiency and restricted dissemination of products. While biomembranes provide structure and define the boundaries of the cell, their dynamic biochemical and biophysical properties also allow them to regulate traffic and communication to and from the cytosol, organize reaction sequences and promote cellular processes. This makes biological membranes and their predominant components, lipids, central constituents of both prokaryotic and eukaryotic cells, responsible for fundamental processes such as energy transduction, cell-to-cell communication, cell division and maintenance of homeostasis.1 Thus, the
biochemical and biophysical study of membranes will ultimately contribute to a better understanding of not just membrane dynamics but of cell function as a whole.
1.1.1. Biomembrane Structure
The key structural feature, shared by all biomembranes, was first described by Gorter and Grendel in 1925.2 While studying lipid extracts of human erythrocytes and depositing them as lipid
monolayers, they observed that the monolayers occupied twice the area than that of the erythrocyte surface area. This led them to propose the lipid bilayer, with the polar headgroups pointing toward the aqueous environment, as the structural foundation of the plasma membrane. In 1935, Danielli and Davson introduced the earliest molecular model of the membrane that included proteins adsorbed to the surface of either side of the membrane, in an attempt to explain selective transport of ions and apolar molecules.3 These two models were later extended to the membranes of different organelles with the
development of electron microscopy.4
These concepts, alongside several studies of permeability and diffusion of lipids and proteins within membranes, were fundamental for the development of the famous fluid mosaic model, proposed by Singer and Nicolson in 1972.5 In the fluid mosaic model, biomembranes are depicted as a matrix
that consists of a fluid bilayer of phospholipids with freely and randomly diffusing globular integral membrane proteins and glycoproteins intercalated within the fluid bilayer. In this model, although a small fraction of the lipid could interact with membrane proteins, lipid-lipid interactions formed the thermodynamic basis for the structural membrane organization. The structure therefore being analogous to a two-dimensional solution of integral proteins and glycoproteins in a phospholipid bilayer solvent.5
Although the basic premises of the fluid mosaic model are still used to explain biomembranes, over the following years it was found that it did not provide adequate explanations for the dynamics of every membrane structure or the great diversity of lipids found in the cell.6 Along the years, the fluid
mosaic model had to be updated to take into account new findings that arose from the implementation of newly developed techniques. These findings include the transversal and lateral asymmetry of lipid distribution, the segregation of certain lipids and proteins into membrane domains, interactions with the cytoskeleton and especially the raft hypothesis.
The raft hypothesis predicts the existence of small (10-200nm), highly dynamic, sterol/sphingolipid enriched domains that can compartmentalize cellular processes.7 It is thought that
4
predicts that certain lipids and proteins, transiently segregate into highly concentrated nanodomains with specific cell functions (e.g. signalling, trafficking).9 The basis for the stabilization of these “temporary
platforms”, resides in specific lipid-lipid, protein-protein and protein-lipid interactions.7 For a full
understanding of these domains, however, one must first consider the different biochemical and biophysical properties of each lipid and protein involved. 10
With this in mind, it is now recognized that the randomness implied in the fluid mosaic model does not exist, as fluid membranes impose a non-random lateral organization of components. The presence of structured lipid domains, especially, complement the original view of the membrane by organizing and ordering many different molecular species through specific interactions, creating membrane compartments that support many aspects of membrane and cellular function (Figure 1.1). 11
1.2. Phosphoinositides
Phosphoinositides (PIs) are a small group of glycerophospholipids derived from phosphatidylinositol (PtdIns). These lipids contain a characteristic inositol headgroup, containing 5 hydroxilations, three of which (at positions 3, 4 and 5) can undergo reversible phosphorylation and dephosphorylation, in all combinations and configurations by several lipid kinases and phosphatases (Figure 1.2 A). 12 This leads to the formation of seven distinct PIs that account for around 2-3% of the
total membrane phospholipids of the eukaryotic cell 13, with PI(4)P and PI(4,5)P
2 (Figure 1.2 B)
representing the bulk of these lipids. 14
Each of these seven species has a distinct subcellular distribution with a predominant localization in subsets of membranes. Additionally, within a given membrane the localization of a specific PI can be heterogeneous. Many PIs are overall in low abundance in the membrane but they can be found at high local concentrations in membrane domains not readily detected by conventional techniques. 14,15
Figure 1.1 An updated view of the fluid-mosaic model. It includes membrane domain structures, interactions with the
5 Despite their low concentration in biomembranes, PIs play an essential role in the recruitment and/or activation of effector proteins and as the precursors of secondary messengers generated by phospholipases. PIs are crucial in cellular processes such as endocytosis/exocytosis, vesicular transport, cytoskeleton regulation, cell growth and cell polarization.16 Furthermore, they have been implicated in
several human diseases like cancer, Lowe syndrome, myotubular myopathy and Charcot Marie Tooth disease. 14,17
Structurally, PIs consist of glycerol esterified in positions sn1 and sn2 by two fatty acid chains and linked to an inositol ring by a phosphodiester bond in position sn3. The most common fatty acids in human (50-80%) are stearic acid (18:0) and arachidonic acid (20:4) in positions sn1 and sn2, respectively, but other minor molecular species are known to coexist in most cells (Figure 1.2B). 18 This
fatty acid profile is uniform across the different PI species, suggesting that most PIs act as a single rapidly interconverting metabolic pool.19
1.3. PI(4,5)P2
PI(4,5)P2 is the most abundant phosphoinositide in mammalian cells and is found primarily in
the inner leaflet of the plasma membrane. It has also been found in endosomes, in the endoplasmic reticulum and in the nucleus.13 Due to two phosphorylations at positions 4 and 5 of the inositol ring it
presents a large negatively charged headgroup, with an expected charge of -4 at pH 7.00. 20 It is worth
noting, however, that the net charge is influenced by several factors such as ionic strength of the medium and interaction with proteins. 21
Because of its biophysical properties, PI(4,5)P2 has the ability to interact, recruit and/or regulate
a variety of signalling proteins. It plays a central role in a spectrum of plasma membrane events, including cell adhesion and motility14, vesicle endocytosis22–24 and exocytosis23,25,26 and ion channel
transport27 (Figure 1.3).
A
B
Figure 1.2 PI molecular structures. (A) General phosphoinositide family structure. Positions 3, 4 and 5 of the inositol ring are
able to undergo phosphorylation. (B) Predominant structure of PI(4,5)P2. It is phosphorylated in positions 4 and 5 of the inositol ring. The most common fatty acids found in human are represented, with stearic acid (18:0) and arachidonic acid (20:4) in positions sn1 and sn2, respectively.
6
1.3.1. PI(4,5)P2 metabolism
PI(4,5)P2 levels are highly regulated by kinases and phosphatases. In human, the majority of
PI(4,5)P2 is synthesized from PI(4)P by type I PIP5Ks (α, β and γ) localized at the plasma membrane. 28
Type II PIP4K (α, β and γ) phosphorylate PI(5)P2 to synthesize a quantitatively minor pool of PI(4,5)P2
localized in the Golgi. 13,29,30 It can also be produced by the dephosphorylation of PI(3,4,5)P
3 catalysed
by PTEN and TPIP (α, β and γ). 18,31
PI(4,5)P2 hydrolysis is controlled by specific 4’- or 5’-phosphatases or by phospholipase
breakdown in response to various stimuli. Dephosphorilation, primarily by 5’-phosphatases, controls PI(4,5)P2 steady-state levels and controls the extent of its signalling. 5’-Phosphatases include the
synaptojanins and the OCRL protein. The synaptojanins function in synaptic vesicle exocytosis and recycling and were found to be critical in determining the fate of the endocytosed clathrin coated vesicles, while the OCRL protein, when mutated, has been found responsible for Lowe Syndrome. 15,32
Cleavage by phospholipases, such as phospholipase C (PLC), control PI(4,5)P2 levels and
originate metabolites that propagate and amplify cellular signalling. PLC enzymes are a key component of receptor-regulated signal transduction. Eukaryotic PI specific PLCs hydrolyse the phosphodiester bond at the position sn3 of phosphoinositides. These PLCs act primarily on PI(4,5)P2 originating the key
secondary messengers Ins(1,4,5)P3 and diacylglycerol (DAG).15
PI(4,5)P2 can also be converted to PI(3,4,5)P3 primarily by class Ia PI3Kinases downstream of
growth factor stimulation. PI(3,4,5)P3 is another crucial secondary messenger that plays a major role in
the regulation of cell proliferation, migration, differentiation and survival. 14
1.3.2. PI(4,5)P2 lateral organization
PI(4,5)P2 is engaged in a multitude of cellular functions occurring in parallel, and the levels of
PI(4,5)P2 are tightly regulated to avoid significant fluctuations of its total plasma membrane
concentration. This suggests that the simultaneous regulation of these cellular functions by PI(4,5)P2
must occur through the presence of different pools of this phospholipid in the plasma membrane,
7 possibly established through protein interactions and a non-homogeneous distribution of PI(4,5)P2 in
the plasma membrane. 21
PI(4,5)P2 lateral organization in cells has been studied through a variety of techniques from
Fluorescence correlation spectroscopy (FCS) and Fluorescence recovery after photobleaching (FRAP) to Atomic force microscopy (AFM). In FCS experiments carried out in Rat1 fibroblasts and HEK cells, researchers microinjected micelles of fluorescent labeled-PI(4,5)P2 into cells and showed that the
diffusion coefficient of PI(4,5)P2 in these cells is significantly lower than in model membranes. The
simplest interpretation of this result is that approximately two thirds of PI(4,5)P2 in the inner leaflet of
the plasma membrane is somehow sequestered. 33 Studies in PC12 cells have also shown, using
Stimulated emission depletion (STED)34 microscopy and Stochastic optical reconstruction microscopy
(STORM)35 imaging techniques, that PI(4,5)P
2 clusters in nanometre sized membrane domains specific
to this cellular model.
While the presence of PI(4,5)P2 pools can be partly explained by localized PI(4,5)P2 synthesis
and degradation through several kinases and phosphatases36, it is also evident that membrane diffusion
rates, in the absence of significant obstacles for diffusion, will always be higher than concentration changes due to enzymatic activity, causing PI(4,5)P2 to diffuse away faster than it can be produced.
This means that it is unlikely that local synthesis can result in significant changes in the submicroscopic organization of PI(4,5)P2 inthe membrane. 21 Protein sequestration and electrostatic interactions could
alternatively explain the observed lateral organization of this phosphoinositide.
1.3.3. PI(4,5)P2 sequestration by proteins
One way to explain PI(4,5)P2 lateral organization in the plasma membrane of cells, is that
proteins can act as reversible buffers, binding much of the PI(4,5)P2 present and then releasing it locally
in response to specific signals.37 Due to its highly negatively charged headgroup, PI(4,5)P
2 can interact
strongly with polybasic stretches of aminoacid residues. 16,21 Through these polybasic stretches, several
proteins, were found to laterally sequester PI(4,5)P2 molecules in a reversible manner. 38,39 For an
efficient buffering of PI(4,5)P2 levels, these proteins would have to be present at a concentration
comparable to PI(4,5)P2, localize to the plasma membrane and be able to bind PI(4,5)P2 with high
affinity while being able to release it in response to stimuli.
Proteins such as MARCKS39,40,41, GAP4337,42, CAP2337 and syntaxin-1A34 have been shown to
be able to sequester PI(4,5)P2 in such a manner. In fact, results show that MARCKS alone could
reversibly sequester much of the PI(4,5)P2 in the plasma membrane. 39 MARCKS sequestration of
PI(4,5)P2 has been shown to be important in the PI(4,5)P2 mediated activation of TRPC-family Ca
channels43, in the endocytosis of the amyloid precursor protein (APP)44 and in the synaptic clustering of
PI(4,5)P2.45
1.3.4. PI(4,5)P2 and divalent cations
PIs and PI(4,5)P2 are able to establish strong electrostatic interactions between their negatively
charged headgroups and positively charged molecules or divalent cations. It has been shown through different techniques that divalent cations, and especially Ca2+,are able to induce the formation of
8
PI(4,5)P2 microdomains. In lipid monolayers, these clusters can be detected through AFM, even in
physiological conditions.46 Recently, it was also shown that the incorporation of PI(4,5)P
2 fluorescent
analogues on PI(4,5)P2 clusters in free-standing lipid bilayers can be detected at fully physiological
conditions using fluorescence spectroscopy methodologies.47
Molecular dynamics (MD) simulation data results show that the ability of divalent cations to promote PI(4,5)P2 cluster formation decreases with increasing counterion sphere size.48 This is
confirmed the several experimental results that suggest that magnesium is weaker than calcium in forming clusters.46,49 In this way, although calcium and magnesium interact with PI(4,5)P
2 in a similar
manner, significantly higher concentrations of magnesium are required to achieve the same level of PI(4,5)P2 clustering as with calcium. In classical simulations, it was found that calcium and magnesium
ions behave similarly until 6Å away from the lipid, at which point the excess free energy necessary to desolvate the hydration shell of magnesium prevents it from coming closer to PI(4,5)P2.50
Furthermore, divalent cations have also shown to be able to influence PI(4,5)P2 electrostatics,
via counterion accumulation (where divalent cations are shown to be more effective than monovalent cations), leading to a charge shielding effect.51 Another important effect, is the influence of divalent
cations, mainly calcium, on the PI(4,5)P2 headgroup conformation, via molecular interactions with the
phosphates as well as the deeply hidden carbonyl groups.52 These effects can heavily influence and
regulate PI(4,5)P2 interactions with binding proteins.
Calcium is a common player in signal transduction and a second messenger in cells. Its levels are strictly controlled and maintained at low levels in the cytosol, with normal intracellular levels at around 100 nM (20 000 fold lower than extracellular levels).53 Upon stimulation, however, several signal
transduction pathways can lead to an increase of intracellular calcium concentration up to around 1 μM, with local concentrations in the vicinity of open calcium channels reaching hundreds of μM.54 In fact,
PI(4,5)P2 has been reported to be associated with a variety of Ca2+ channels and a great number of these
require PI(4,5)P2 for proper function.15
Magnesium, a less studied crucial modulator of cell function, has its cellular levels well buffered in a narrow millimolar range between 0.25 mM and 1 mM.55,56 Interestingly, magnesium levels are kept
at a much higher concentration than those of calcium, which might compensate the lower sensitivity presented by PI(4,5)P2 towards this cation.
Altogether, this suggests that it is possible that divalent cationmediated clustering leads to the formation of specific sites in the membrane highly enriched in PI(4,5)P2, while depleting the rest of the
membrane.47 These cation-induced effects can influence not only PI(4,5)P
2 lateral organization but also
the way PI(4,5)P2 interacts with proteins, by modulating their localization in the plasma membrane,
their target recognition and binding affinity to PI(4,5)P2, and even further interactions with other
proteins. It is highly likely that the interactions of divalent cations with PI(4,5)P2 have a crucial role in
the regulation of the biological activity of this phospholipid.
In this study, we will focus on the impact of calcium on PI(4,5)P2, as it has the highest affinity
towards the phospholipid. However, physiologically, magnesium and calcium most likely act in concert,
9
1.3.5. PI(4,5)P2 and protein interactions
PI(4,5)P2 participates in signalling events through binding of its headgroup to a multitude of
different lipid binding protein domains (Table 1.1). PI(4,5)P2 has a large headgroup that may protrude
further into the aqueous phase than a typical phospholipid and presents a highly negative charge density, which stands out in the inner leaflet of the plasma membrane, allowing recognition and binding through either unstructured basic residue-rich regions or more structured protein domains. 21
Binding of PI(4,5)P2 to lipid binding domains can serve to activate proteins as well as merely
anchoring them to the plasma membrane. The pleckstrin homology (PH) domain of PLCδ contributes to its targeting towards the inner leaflet of the plasma membrane versus internal membranes, while the C1 and C2 domains of PKC have a role in its activity besides membrane targeting. 21
These lipid binding domains have distinct structures and can bind by a combination of stereochemical recognition of the headgroup and by nonspecific long range electrostatic or hydrophobic interactions, leading to different specificities and affinities. In this thesis, we aim to study the impact of calcium on the affinity of PI(4,5)P2 for different PI(4,5)P2 binding proteins and on their membrane
organization. To this end, we chose a protein domain where stereochemical recognition plays a crucial role in binding, the PH domain, and a smaller peptide for which binding is apparently uniquely governed by electrostatic contributions, the polyphosphoinositide-binding peptide (PBP). Finally we also want to characterize the impact of calcium, in a more complex lipid-protein system, namely the association between PI(4,5)P2 and HIV-1 Gag precursor protein, for which in addition to these previously studied
factors, membrane composition also plays a role in governing PI(4,5)P2-protein organization.
Surprisingly, the impact of divalent cations on the interaction of PI(4,5)P2 with these proteins
has been given little attention up until recently, where a new surge of interest emerged. Divalent cations could be found to act as an additional regulatory mechanism of PI(4,5)P2 – protein affinity and even of
PI(4,5)P2 binding protein organization.
Table 1.1 Representative PI(4,5)P2 interacting domains and proteins. Adapted from [15,57].
Interacting Domain
Proteins
References
PH Dynamin, PLC-δ [58,59]
ENTH Epsin1 [60]
ANTH AP18D/CALM [61]
PX SNX9, CPK PI3K, TCGAP [62,63,64]
FERM Ezrin, Radixin, PTPL1 [65,66, 67, 68]
C2 Synaptotagmin-1 [69]
10
1.3.5.1. Pleckstrin homology (PH) domains
The pleckstrin homology (PH) domain consists of a 100-120 residue long amino acid sequence found in numerous proteins involved in cellular signalling. PH domains direct membrane targeting of their host proteins by binding to phosphoinositides. Of the total number of PH domains detected in protein sequences, only a small minority bind specifically to PIs. Most bind weakly and non-specifically.74
Each characterized PH domain has essentially the same structure, consisting of a β sandwich closed off at one end by a C-terminal α helix 75, with the splayed corner of the β sandwich containing 3
variable loops, that have been suggested, by analogy with immunoglobulin-like domains, to be the binding site (Figure 1.4).74 It has also been found that PH domains are electrostatically polarized with
the positively charged end coinciding with the three variable loops. It is through this region that the PH domain binds to the polyphosphorylated inositol ring. 76
The isolated PLCδ1 PH domain has been found to bind with high affinity and specificity to PI(4,5)P2 and its soluble headgroup Ins(1,4,5)P3 . 77 It contains two additional short α helices not found
in other PH domains but neither interfere in the binding to the phosphorylated inositol headgroup. Through direct hydrogen bonds between the bound headgroup and seven amino acid residues, the 4- and 5- phosphate groups of the phosphorylated inositol headgroup are clamped and buried in the binding pocket. These interactions suggest that stereochemical cooperativity enhance specificity. 78
It is worth noting that while PLCδ1PH binds to PI(4,5)P2 with high affinity, FRAP results
suggest that its steady-state membrane localization is the result of its very rapid cycling between a membrane bound and a cytosolic state, with on-off rates in the range of seconds.79 Also
microdissociation and rebinding events have been detected for PH domains in single molecule studies, confirming a transient binding mechanism. 80
A
B
Figure 1.4 PH domain 3D protein structure examples. (A) PDK1 PH domain bound to DIC4-Ins(3,4,5)P3. (PDB Entry: 1W1G) 178 (B) PLCδ1 PH domain bound to Ins(1,4,5)P3. (PDB Entry: 1MAI). 78 Molecular graphics were performed with the UCSF Chimera package. 164
11
1.3.5.2. Polyphosphoinositide-binding peptide, PBP-10
Polyphosphoinositide-binding peptide (PBP-10) is a 10 residue long, membrane-permeant polycationic peptide derived from the PI(4,5)P2 binding region in segment-2 of gelsolin (Gelsolin
residues 160-169) which was linked to rhodamine B (RhB-QRLFQVKGRR).81,82
Gelsolin is an 82kDa protein with an important role in the regulation of cytoskeleton dynamics through the severing and capping of actin filaments. It is one of the most potent actin filament severing proteins identified, severing stoichiometrically with nearly 100% efficiency. 83 Gelsolin is regulated by
fluctuations in Ca2+ levels and by interaction with PIs, especially the tight and specific binding to
PI(4,5)P2.83 The 10 amino acid residue sequence of PBP-10 (Figure 1.5) is one of two identified
responsible for the PI binding activity of intact gelsolin. 81 Sequences similar to these have been found
in other PI binding proteins, such as phospholipase Cβ.81,84
PBP-10 is a polyvalent cation with several hydrophobic residues, that similarly to the intact protein, binds avidly to PIs. It’s binding to PIs appears to be governed mostly by electrostatics, however, it has been suggested that it has a steric contribution, since it shows much higher affinity than that of similar peptides with higher positive charges. 81 Unlike the intact protein, PBP-10 appears to be much
less selective towards different PIs, binding with similar strength towards PI(3,4)P2 and PI(4,5)P2. 81
Upon binding to membranes containing acidic lipids, PBP-10 undergoes a strong reduction in fluorescence intensity. 81,82 This gradual loss of fluorescence intensity, observed with the increase in
total lipid concentration, is likely caused by the self-quenching of rhodamine B groups as the peptide oligomerizes 82 at the vesicle surface after membrane association.
12
1.3.5.3. Influence of Ca2+ in PI(4,5)P
2-protein and protein-protein interactions
Many PI(4,5)P2 binding proteins are known to be sensitive to variations in Ca2+ concentration.
In most of these cases, calcium causes changes in protein folding/electrostatics that affect the affinity of PI(4,5)P2 binding proteins leading to an increase of binding to the membrane. In these proteins, PI(4,5)P2
and calcium binding typically occurs at different binding sites. 85 It is also worth noting that the
formation of PI(4,5)P2 clusters due to divalent cation cross-linking, as described previously, is likely to
have a dramatic effect not only on protein targeting or activity but also on the promotion/inhibition of protein-protein interactions.
Several signalling proteins containing C2 domains, such as rabphilin-3A86, protein kinase C
(PKC)87 or synaptotagmin88, have their affinity for PI(4,5)P
2 regulated in a Ca2+ -dependent manner.
Binding of several annexins to PI(4,5)P2 has also been shown to be calcium concentration dependent 85,
where it can even lead to the formation of micrometre sized PI(4,5)P2 clusters, as is the case of annexin
A2t.89 Syntaxin, a SNARE protein that catalyses regulated exocytosis, has also been found to cluster
with PI(4,5)P2 in the plasma membrane, through an unknown mechanism promoted by physiological
concentrations of calcium. 90
In the presence of calcium, calmodulin (CaM) competes with PI(4,5)P2 for binding to several
CaM protein targets that were shown to also bind to PI(4,5)P2, regulating the association/dissociation of
these proteins from the plasma membrane. 16
1.4. Human Immunodeficiency virus type 1 (HIV-1)
HIV-1 is the causative agent of acquired immune deficiency syndrome (AIDS), one of the most dangerous viral pandemics in history. HIV infection is characterized by an acute phase of intense viral replication and dissemination to lymphoid tissues; a chronic, often asymptomatic phase of sustained immune activation and viral replication; and ultimately an advanced phase of marked depletion of CD4+
T cells that leads to AIDS. This weakens the immune system and allows for an entire spectrum of opportunistic infections and pathological conditions. 91 HIV research and the continuous development
of approaches to tackle HIV infection are crucial to explore novel therapeutic strategies against this pandemic.
HIV-1 is classified in a separate genus of the Retroviridae family called the lentiviridae, based on its morphological, genetic and biological properties. 92 Lentiviruses are known for their cytolytic and
immunosuppressive properties as well as their complex genomes, that contain accessory and regulatory genes. 93
1.4.1. HIV-1 replication cycle
The HIV-1 replication cycle can be divided into an early and a late phase (Figure 1.6). The early phase encompasses the events that occur from the virus binding to the surface of the host CD4+ T
13 cell until the integration of viral DNA into the host genome. During the early phase, direct fusion of the virion and cellular lipid membranes occurs, the viral core is released into the cytoplasm where it uncoats, releasing the viral RNA genome. The viral genome is then reverse transcribed and transported to the nucleus where it integrates the hosts genome as a provirus. Most therapeutic solutions currently implemented block viral replication processes in the early phase of the cycle94.
The late phase includes the events from the transcription of viral genes, the assembly, release and maturation of new virions. 93,95 During the late phase, the viral genes are transcribed and the viral
RNA exported to the cytosol of the host cell where it is translated, forming the viral proteins. These proteins include Gag, a 55kDa precursor protein that forms the viral particle and GagPol, a 160kDa polyprotein that contains the viral protease, reverse transcriptase and integrase, expressed at an approximate 5% of the level of Gag. Gag, GagPol and the envelope glycoproteins are trafficked to the plasma membrane where the assembly of Gag and Gagpol occurs. This leads to the formation of the capsid, encapsidation of the viral RNA, incorporation of the envelope glycoproteins and finally budding off of the virions and particle maturation. 95
1.4.2. Gag precursor protein
HIV-1 Gag is a 55kDa polyprotein that contains four domains (Figure 1.7): the matrix domain (MA); the capsid domain (CA); the nucleocapsid domain (NC); and the p6 domain. It also includes two small spacer peptides, SP1 and SP2. (Figure 1.5) As the structural protein of HIV-1, Gag alone drives the assembly process of the virus particle, where each of its domains performs a specific and distinct function during the process. 96,97,98
The N-terminal MA domain is responsible for the directing and anchoring of Gag to the cell membrane. The Central CA domain, promotes homo-oligomerization in an ordered manner during the
Figure 1.6 HIV-1 replication cycle. The main steps are illustrated, as well as the major antiretroviral drugs next to the step of
14
assembly and is the critical determinant of particle morphology. The NC domain binds RNA, leading to its encapsidation while also promoting oligomerization. P6, the C-terminal domain, is largely unstructured but contains the docking sites for the ESCRT and ESCRT-associated proteins that are required for the scission of the virion envelope from the host plasma membrane. 98
1.4.2.1. Gag membrane targeting and binding
The Gag MA domain contains the entities that allow Gag membrane targeting and binding: the N-terminal myristoyl moiety modification and a highly basic region (HBR) that comprises residues 17-31. 99
The myristate moiety is essential for Gag membrane binding and virus release in cells, as proved by experiments with myristoyl-deficient Gag proteins. 100,101 In addition, it has also been shown that the
myristoyl moiety may have a role in Gag-Gag multimerization.102 In Gag, as in many other myristoylated
proteins103–105, the myristate moiety can either be sequestered within the molecule or exposed to the
surrounding environment.102 The transition between these two states has been coined myristoyl switch. 106 In this case, the myristoyl moiety is sequestered within the hydrophobic cleft of the MA domain.
Even though the myristoylation is necessary for Gag binding, it is thought to be insufficient for efficient binding and targeting as it only provides reversible membrane binding.106,107 The HBR acts as
a second signal, crucial for strong localization of the protein to the plasma membrane and efficient membrane binding through the interaction with acidic lipids. 99 Mutations in the MA domain HBR lead
to mislocalization of Gag to intracellular compartments and reduced Gag membrane binding. 99,108,109 Figure 1.7 HIV-1 Gag protein structure and associated function. Adapted from [95].
15
1.4.2.2. Gag interaction with PI(4,5)P2
HBR is known to interact with lipids such as PS or PIs. Amongst the PIs, PI(4,5)P2 is considered
to be the more specific110,111 but PI(3,4)P
2 or PI(3,4,5)P2 also bind efficiently.112 As is the case for other
poly-basic proteins, the initial attractive force towards the membrane occurs at long range distances and is mainly due to electrostatic interaction between the plasma membrane and the Gag protein HBR.
PI(4,5)P2 is critical for HIV-1 assembly, as shown, on a study where cellular PI(4,5)P2 is
depleted in HeLa cells using a specific phosphatase (5ptaseIV). This led to Gag mislocalization to internal compartments and significantly reduced virion release. 113 In vitro studies have also shown the
need for PIs to obtain normal HIV-1 particle assembly. 114 An additional level of regulation of Gag
targeting to the membrane, promoted specifically by PI(4,5)P2,was suggested after the observation that
RNA inhibits Gag-membrane interactions by binding to the HBR. PI(4,5)P2 but not other acidic
phospholipids, were found to be able to outcompete RNA for MA binding. 115,116
Recently, PI(4,5)P2 interactions with Gag have been heavily studied through NMR and
simulation techniques. NMR studies of HIV-1 MA domain bound to diC4-PI(4,5)P2 or diC 8- PI(4,5)P2
have shown that PI(4,5)P2 can induce a myristoyl switch, from the hydrophobic pocket in the MA into
the membrane, through electrostatic binding. 117–119 It has also been found that the myristoyl switch can
occur spontaneously in Gag molecules in solution, with free energy values suggesting an equilibrium between the sequestered and exposed conformation. However, the myristoyl moiety remains most of the time at the protein surface to prevent contacts with the surrounding water molecules.120 This is an
interesting find, as previously, it was suggested that Gag released its myristoyl moiety and probed the membrane until finding PI(4,5)P2 and establishing stable membrane binding.121 Nowadays, it has been
suggested, taking into account NMR and simulation studies, that myristate insertion occurs after non-specific attraction to the membrane and HBR capture of the phosphorylated inositol head group of PI(4,5)P2. This myristate insertion is fundamental for the favourable position of the HBR for specific
interactions with PI(4,5)P2 . 120
Figure 1.8 Membrane binding model of MA:PS:PI(4,5)P2. The tripartite engagement shows how the MA is anchored to the
plasma membrane via the sn1 acyl chains of PI(4,5)P2 and PS and the myristate moiety whilst the SN2 acyl chains are buried deep within the hydrophobic cavities. Adapted from [122].
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Structural studies have also shown that PI(4,5)P2 can adopt an “extended lipid” conformation
when binding to Gag. In this conformation, the inositol head group interacts with HBR and the PI(4,5)P2
long typically unsaturated acyl chain in position sn2 is sequestered in a hydrophobic cluster of MA concomitantly to the myristate switch. Meanwhile Gag holds on to the membrane through the typically saturated sn1 acyl chain and the myristate moiety. 117 New NMR data shows the existence in the MA
domain of a second lipid binding site inducing sn2 acyl chain flipping into a hydrophobic pocket. In this case there isn’t a specific bound lipid and it can be PS, PC or PE. These models seem to indicate that the MA is able to localy deplete the bound lipids of unsaturated chains. 122
A recent study has examined Gag binding to membranes containing acyl chain variants of PI(4,5)P2. It was found that the acyl chains of PI(4,5)P2 play an important role in Gag binding to
membranes as brain-PI(4,5)P2 and DO-PI(4,5)P2 but not DP-PI(4,5)P2 supported Gag binding to
membranes. This suggests that unsaturated acyl chains are necessary for PI(4,5)P2 dependent Gag
retention. This effect was observed even for RNase-treated, nonmyristoylated Gag suggesting that the MA domain determines Gag preference for PI(4,5)P2 with unsaturated acyl chains and not through
interactions with the myristate moiety. This could mean that Gag is only able to sequester unsaturated acyl chains in its core as a saturated acyl chain may encounter a steric hindrance or an energetic barrier.123 Thus, Gag appears to effectively have a tripartite membrane interaction (Figure 1.8) consisting
of the N-terminal myristate, the HBR and the hydrophobic grooves that bind the flipped out acyl chains.
1.4.2.3. Role of lipid domains in the interaction of Gag with the plasma membrane
The effect of lipid domains in Gag interaction with the membrane has been studied extensively. HIV-1 acquires its lipid envelope and envelope proteins by Gag assembly and budding at the plasma membrane of the infected cells, therefore the envelope composition reflects some of the interaction between Gag and the plasma membrane.124 While the enrichment in the envelope seems to be dependent
on the membrane of the host cells, enrichment of several “raft lipids” has been observed.125–127
Lipidomics studies have shown that the HIV-1 envelope is enriched in PIs125,128 but also in
sphingomyelin.128 Enrichment in cholesterol, PS, PE 125,128 and an overall increase in saturated fatty
acids compared with the host cells has also been reported128. While there is almost no doubt that the
envelope is enriched in these lipids, it is not yet clear at which step of the assembly this enrichment occurs. 129
Gag could either target pre-existing inner leaflet domains or lead to assembly-induced lipid domains. However, assembly-induced lipid domains seem the most likely129 as several membrane
binding proteins and cytoskeleton interactions have been shown to induce lipid phase separation.130
Also, coarse grained molecular dynamics simulation of the interaction of the MA domain with the inner leaflet of the plasma membrane have shown a potential for enrichment in PI(4,5)P2 and acidic lipids. 120
This data, combined with the depletion of unsaturated chains from the Gag bound lipids, discussed previously, could create a local environment that may be driving the assembly of these lipid domains. Whatever the origin of this enrichment, it’s role in favouring virus assembly is yet unknown and a target of discussion. 129