Biology subjects

Kalli, A. C.

Publications and source records attributed to Kalli, A. C..

15 recordsLinked to original sources

bioRxiv2023

Rapid Prediction of Lipid Interaction Sites on Pleckstrin Homology Domains Using Deep Graph Neural Networks and Molecular Dynamics Simulations

Interactions between membrane proteins and specific lipid molecules play a major role in cellular biology, but characterizing these interactions can be challenging due to the complexity and physicochemical properties of membranes. Molecular dynamics (MD) simulations allow researchers to predict protein-lipid interaction sites and generate testable models. MD simulations are however computationally expensive and require specialist expertise. In this study, we demonstrate that graph neural networks trained on coarse-grained MD simulation data can predict phosphoinositide lipid interaction sites on Pleckstrin Homology (PH) domain structures, a large family of membrane binding domains. The predictions are comparable to the results of simulations and require only seconds to compute. Comparison with experimental data shows that the model can predict known phosphoinositide interaction sites and can be used to form hypotheses for PH domains for which there is no experimental data. This model is a next generation tool for predicting protein-lipid interactions of PH domains and offers a basis for further development of models applicable to other membrane protein classes.

biophysics↗

bioRxiv2023

A molecular switch in RCK2 triggers sodium-dependent activation of KNa1.1 (KCNT1) potassium channels.

The Na+-activated K+ channel KNa1.1, encoded by the KCNT1 gene, is an important regulator of neuronal excitability. How intracellular Na+ ions bind and increase channel activity is not well understood. Analysis of KNa1.1 channel structures indicate that there is a large twisting of the {beta}N-Q loop in the intracellular RCK2 domain between the inactive and Na+-activated conformations, with a lysine (K885, human subunit numbering) close enough to form a salt bridge with aspartate (D839) in the Na+-activated state. Concurrently, an aspartate (D884) adjacent in the same loop adopts a position within 4 [A] of several acidic or polar residues. In carrying out mutagenesis and electrophysiology with human KNa1.1, we found alanine substitution of each of these residues resulted in almost negligible currents in the presence of up to 40 mM intracellular Na+. The exception was D884A, which resulted in constitutively active channels in both the presence and absence of intracellular Na+. Further mutagenesis of this site revealed an amino acid size-dependent effect. Substitutions at this site by an amino acid smaller than aspartate (D884V) also yielded constitutively active KNa1.1, D884I had Na+- dependence similar to wild-type KNa1.1, whilst increasing the side chain size larger than aspartate (D884E or D884F) yielded channels that could not be activated by up to 40 mM intracellular Na+. We conclude that Na+ binding results in a conformational change that accommodates D884 in the acid-rich pocket, which triggers further conformational changes in the RCK domains and channel activation. Statement of SignificanceSodium-activated potassium channels regulate neuronal excitability, and their dysfunction causes severe childhood disorders. Here, we identify a structural determinant in the intracellular domains that is responsible for triggering channel activation in response to sodium ion binding. An increase in the size of a particular amino acid renders the channel sodium-insensitive, whilst a decrease in size enables the channel to activate in the absence of sodium. This enhances our understanding of how this subclass of potassium channels respond to changes in the intracellular ionic environment. Furthermore, this may also further our understanding of the basis of human neurological disorders and their treatment.

biophysics↗

bioRxiv2022

Molecular dynamics simulations reveal membrane lipid interactions of the full-length lymphocyte specific kinase Lck

The membrane-bound lymphocyte-specific protein-tyrosine kinase (Lck) triggers T cell antigen receptor signalling to initiate adaptive immune responses. Despite many structure-function studies, the mode of action of Lck and the potential role of plasma membrane lipids in regulating Lcks activity remains elusive. Advances in molecular dynamics simulations of membrane proteins in complex lipid bilayers have opened a new perspective in gathering such information. Here, we have modelled the full-length Lck open and closed conformations available from crystallographic studies and simulated its interaction with the inner leaflet of the T cell plasma membrane. In both conformations, we found that the unstructured unique domain and the structured domains including the kinase interacted with the membrane with a preference for PIP lipids. Interestingly, our simulations suggest that the Lck-SH2 domain interacts with lipids differently in the open and closed Lck conformations, demonstrating that lipid interaction can potentially regulate Lcks conformation and in turn modulate T cell signalling. Additionally, the Lck-SH2 and kinase domain residues that significantly contacted PIP lipids are found to be conserved among the Src family of kinases, thereby potentially representing similar PIP interactions within the family.

biophysics↗

bioRxiv2022

mPPases create a conserved anionic membrane fingerprint as identified via multi-scale simulations

Membrane-integral pyrophosphatases (mPPases) are membrane-bound enzymes responsible for hydrolysing inorganic pyrophosphate and translocating a cation across the membrane. Their function is essential for the infectivity of clinically relevant protozoan parasites and plant maturation. Recent developments have indicated that their mechanism is more complicated than previously thought and that the membrane environment may be important for their function. In this work, we use multiscale molecular dynamics simulations to demonstrate for the first time that mPPases form specific anionic lipid interactions at 4 sites at the distal and interfacial regions of the protein. These interactions are conserved in simulations of the mPPases from Thermotoga maritima, Vigna radiata and Clostridium leptum and characterised by interactions with positive residues on helices 1, 2, 3 and 4 for the distal site, or 9, 10, 13 and 14 for the interfacial site. Due to the importance of these helices in protein stability and function, these lipid interactions may play a crucial role in the mPPase mechanism and enable future structural and functional studies. Author summaryIn this work we have been able to demonstrate conservation of lipid-interaction sites on proteins from distinct species that deviated from their evolutionary common ancestors a long time ago, as in the case of the membrane-integral pyrophosphatases from a thermophilic bacteria species and a plant species studied here. This retention of a common lipid interaction profile or "fingerprint" and our ability to predict this for other proteins in this family may indicate that they are more integral to protein function than previously thought. By identifying lipid interactions that may act to stabilise the protein structure, these properties could be exploited to gain protein structures, and the interfacial sites potential involvement in inter-subunit communication may be useful for further investigation of the catalytic cycle of this clinically relevant membrane protein family.

biophysics↗

bioRxiv2021

Systematic simulation of the interactions of Pleckstrin homology domains with membranes

Pleckstrin homology (PH) domains can recruit proteins to membranes by recognition of phosphatidylinositol phosphates (PIPs). Here we report the systematic simulation of the interactions of 100 mammalian PH domains with PIP containing model membranes. Comparison with crystal structures of PH domains bound to PIP analogues demonstrates that our method correctly identifies interactions at known canonical and non-canonical sites, while revealing additional functionally important sites for interaction not observed in the crystal structure, such as for P-Rex1 and Akt1. At the family level, we find that the {beta}1 and {beta}2 strands and their connecting loop constitute the primary PIP interaction site for the majority of PH domains, but we highlight interesting exceptional cases. Simultaneous interaction with multiple PIPs and clustering of PIPs induced by PH domain binding are also observed. Our findings support a general paradigm for PH domain membrane association involving multivalent interactions with anionic lipids. TeaserSimulating the binding of 100 Pleckstrin homology domains to cell membranes reveals patterns in their lipid interactions.

biophysics↗

bioRxiv2021

Mechanical and molecular activation lead to structurally analogous MscL states

The MscL channel gates in response to membrane tension changes to allow the exchange of molecules through its pore. Lipid removal from transmembrane pockets leads to a MscL response. However, it is unknown whether there is correlation between the tension mediated state and the state derived by pocket delipidation in the absence of tension. Transitions between MscL states may follow a similar pathway to cover the available conformational space but may not necessarily sample the same discrete intermediates. Here, we combined pulsed-EPR and HDX-MS measurements on MscL, coupled with molecular dynamics under membrane tension, to investigate the changes associated with the distinctively derived states. Whether it is tension or pocket delipidation, we find that MscL samples a similar expanded state, which is the final step of the delipidation pathway but only an intermediate stop of the tension mediated path. Our findings hint at synergistic modes of regulation in mechanosensitive channels.

biophysics↗

bioRxiv2021

Inhibitors of the Small Membrane (M) Protein Viroporin Prevent Zika Virus Infection

Flaviviruses, including Zika virus (ZIKV), are a significant global health concern, yet no licensed antivirals exist to treat disease. The small Membrane (M) protein plays well-defined roles during viral egress and remains within virion membranes following release and maturation. However, it is unclear whether M plays a functional role in this setting. Here, we show that M forms oligomeric membrane-permeabilising channels in vitro, with increased activity at acidic pH and sensitivity to the prototypic channel-blocker, rimantadine. Accordingly, rimantadine blocked an early stage of ZIKV cell culture infection. Structure-based channel models, comprising hexameric arrangements of two trans-membrane domain protomers were shown to comprise more stable assemblages than other oligomers using molecular dynamics (MD) simulations. Models contained a predicted lumenal rimantadine binding site, as well as a second druggable target region on the membrane-exposed periphery. In silico screening enriched for repurposed drugs/compounds predicted to bind to either one site or the other. Hits displayed superior potency in vitro and in cell culture compared with rimantadine, with efficacy demonstrably linked to virion-resident channels. Finally, rimantadine effectively blocked ZIKV viraemia in preclinical models, supporting that M constitutes a physiologically relevant target. This could be explored by repurposing rimantadine, or development of new M-targeted-therapies.

microbiology↗

bioRxiv2021

Multi-scale simulations of the T cell receptor reveal its lipid interactions, dynamics and the arrangement of its cytoplasmic region

The T cell antigen receptor (TCR-CD3) complex initiates T cell activation following recognition of peptides presented by Major Histocompatibility Complex (pMHC)-encoded proteins. The ligation of pMHC to TCR{beta} induces Src family kinases activity via the cytoplasmic tails of the CD3{delta}{varepsilon}, CD3{gamma}{varepsilon} and {zeta}{zeta} dimers. The TCR-CD3 topology is well understood, but little is known about its conformational dynamics and arrangement of its cytoplasmic tails, limiting our grasp of the signalling mechanism. Here, we investigated the entire TCR-CD3 embedded in an asymmetric lipid bilayer using molecular modelling and multi-scale molecular dynamics simulations. Our study demonstrates conformational changes in the extracellular and transmembrane domains, and the arrangement of the TCR-CD3 cytoplasmic tails. The TCR{beta} variable regions were the most flexible in the extracellular domain. The cytoplasmic tails formed highly interlaced structures while some tyrosine sidechains within the immunoreceptor tyrosine-based activation motifs (ITAMs) of the CD3{varepsilon} and {zeta} subunits dynamically penetrated the hydrophobic core of the bilayer. Ionic interactions between the cytoplasmic tails and phosphatidylinositol phosphates (PIP2 and PIP3) in the inner leaflet of the lipid bilayer led to the formation of a distinct annular lipid fingerprint around the TCR-CD3 complex. These results combined with available experiential data increase our understanding of the TCR-CD3 activation mechanism and highlight the importance of membrane lipids in regulating T cell activation. Significance statementThe T cell receptor (TCR-CD3) detects antigenic peptides displayed by major histocompatibility complexes (pMHC) to instigate activation of T cell adaptive immunity. Despite significant structural and functional knowledge of TCR-CD3 topology, the membrane interactions and dynamics of its cytoplasmic moieties remain elusive. Interactions of TCR-CD3 cytoplasmic tails with membrane lipids may regulate their phosphorylation by Src-family kinases, the first intracellular event required for T cell activation. Using the static 3D structure of TCR-CD3 resolved by cryo-electron microscopy, we provide novel insights into the protein-lipid interactions of the complete TCR-CD3 embedded in a bilayer closely mimicking its native membrane environment. Our study sheds light on the dynamics of the TCR-CD3 at near-atomic resolution and further aids in deciphering its activation mechanism.

biophysics↗

bioRxiv2020

Allosteric activation of T cell antigen receptor signalling by quaternary structure relaxation

The mechanism of T cell antigen receptor (TCR-CD3) signalling remains elusive. Here, we identified mutations in the transmembrane region of TCR{beta} or CD3{zeta} that augmented pMHC-induced signalling, not explicable by enhanced ligand binding, lateral diffusion, clustering or co-receptor function. Using a novel biochemical assay and molecular dynamics simulation, we demonstrated that the gain-of-function mutations loosened interaction between TCR{beta} and CD3{zeta}. We found that, similar to the activating mutations, pMHC binding reduced TCR{beta} cohesion with CD3{zeta}. This event occurred prior to CD3{zeta} phosphorylation and at 0{degrees}C. Moreover, we demonstrated that soluble monovalent pMHC alone induced signalling and reduced TCR{beta} cohesion with CD3{zeta} in membrane-bound or solubilised TCR-CD3. Our data provide compelling evidence that pMHC binding suffices to activate allosteric changes propagating from TCR{beta} to the CD3 subunits, reconfiguring interchain transmembrane region interactions. These dynamic modifications could change the arrangement of TCR-CD3 boundary lipids to licence CD3{zeta} phosphorylation and initiate signal propagation.

immunology↗

bioRxiv2020

The guidance and adhesion protein FLRT2 dimerizes in cis via dual Small-X3-Small transmembrane motifs

Fibronectin Leucine-rich Repeat Transmembrane (FLRT 1-3) proteins are a family of broadly expressed single-spanning transmembrane receptors that play key roles in development. Their extracellular domains mediate homotypic cell-cell adhesion and heterotypic protein interactions with other receptors to regulate cell adhesion and guidance. These in trans FLRT interactions determine the formation of signaling complexes of varying complexity and function. Whether FLRTs also interact at the surface of the same cell, in cis, remains unknown. Here, molecular dynamics simulations reveal two dimerization motifs in the FLRT2 transmembrane helix. Single particle tracking experiments show that these Small-X3-Small motifs synergize with a third dimerization motif encoded in the extracellular domain to permit the cis association and co-diffusion patterns of FLRT2 receptors on cells. These results may point to a competitive switching mechanism between in cis and in trans interactions which suggests that homotypic FLRT interaction mirrors the functionalities of classic adhesion molecules. FieldsStructural Biology and Biophysics / Computational Biology

biophysics↗

bioRxiv2019

Inter-domain dynamics in the chaperone SurA and multi-site binding to its unfolded outer membrane protein clients

The periplasmic chaperone SurA plays a key role in outer membrane protein (OMP) biogenesis. E. coli SurA comprises a core domain and two peptidylprolyl isomerase domains (P1 and P2), but how it binds its OMP clients and the mechanism(s) of its chaperone action remain unclear. Here, we have used chemical cross-linking, hydrogen-deuterium exchange, single-molecule FRET and molecular dynamics simulations to map the client binding site(s) on SurA and to interrogate the role of conformational dynamics of the chaperones domains in OMP recognition. We demonstrate that SurA samples a broad array of conformations in solution in which P2 primarily lies closer to the core/P1 domains than suggested by its crystal structure. Multiple binding sites for OMPs are located primarily in the core domain, with binding of the unfolded OMP resulting in conformational changes between the core/P1 domains. Together, the results portray a model in which unfolded OMP substrates bind in a cradle formed between the SurA domains, with structural flexibility between its domains assisting OMP recognition, binding and release.

biochemistry↗

bioRxiv2019

Molecular principles of Piezo1 activation by increased membrane tension

SUMMARY PARAGRAPHPiezo1 is a mechanosensitive channel involved in many cellular functions and responsible for sensing shear-stress and pressure forces in cells1-3. Piezo1 plays a critical role in the circulatory system and tissue development. Mutations on Piezo1 are linked to human diseases such as lymphedema2,4 or hematological disorders such as hemolytic anaemia5 and resistance to malaria6. Hypotheses for Piezo1 gating include the "force-from-lipids" principle7,8 that suggests that Piezo1 senses mechanical forces through the bilayer1,9 and a direct involvement of the cytoskeleton as well as the extracellular matrix in Piezo1 activation10,11. However, the molecular and structural changes underpinning the Piezo1 gating mechanism and how the channel senses forces in the membrane remain unknown. Here we reveal the activation mechanism of Piezo1 and the structural rearrangements that occur when Piezo1 moves from a closed to an open state when mechanical tension is applied to the cell membrane. Our results show that Piezo1s curved shape is stable in a native-like model membrane without tension creating a membrane indentation with a trilobed topology. Upon stretching Piezo1 adapts to the stretched bilayer by flattening and expansion of its blade region. In our simulations Piezo1 expands up to a planar circular area of [~]680 nm2 comparable with previous structural data and hypotheses12-14. Piezo1 flattening and expansion results in changes in the beam helix tilt angle. These movements result in the tilting and lateral movement of the pore lining TM37 and TM38 helices. This leads to the opening of the channel and to the movement of lipids that occupy Piezo1 pore region outside of this region, revealing for the first time the structural changes that happen during Piezo1 mechanical activation. The changes in the blade region are transmitted to helices TM37 and 38 via hydrophobic interactions and by interactions of neighbouring subunits via the elbow region. The flat structure of Piezo1 identified in this study exposes the C-terminal extracellular domain (CED) that in the closed state is hidden in the membrane and presumably from shear stress. Our results provide new structural data for different states of Piezo1 and suggest the molecular principles by which mechanical force opens the Piezo1 channel, thus coupling force to physiological effect via ion permeation.

biophysics↗

bioRxiv2019

Sphingomyelinase disables Piezo1 channel inactivation to enable sustained response to mechanical force

Piezo1 channels are determinants of vascular responses to fluid flow. They importantly provide sustained response to flow, but this property contrasts with the rapid inactivation that has become a hallmark of the channels in heterologous overexpression studies. Here we reveal a mechanism by which blood vessels disable inactivation to enable sustained physiological response. Creation of a molecular model of Piezo1 channel in defined lipid membranes suggested potential modulation by sphingomyelin and its product ceramide. Biological relevance was indicated by the observation that exogenous sphingomyelinase enhanced Piezo1-mediated Ca2+ entry in cultured endothelial cells. We therefore hypothesised that endogenous sphingomyelinase suppresses channel inactivation. Remarkably, in endothelium freshly-isolated from murine artery, neutral sphingomyelinase inhibitors or genetic disruption of sphingomyelin phosphodiesterase 3 (SMPD3) caused flow- and pressure-activated Piezo1 channels to become inactivating. SMPD3 retained its ability to disable inactivation in cell-free membrane patches, providing evidence for a membrane localised effect. The data suggest that inherent inactivation of Piezo1 channels is disabled by enzymatic control of lipid environment to enable physiological response to mechanical force.

physiology↗

bioRxiv2019

Computational reconstruction of the complete Piezo1 structure reveals a unique footprint and specific lipid interactions

Piezo1 is a critical mechanical sensor in many cells. It is activated by mechanical force thus allowing cells to sense the physical environment and respond to stress. Structural data have suggested that Piezo1 has a curved shape. Here, we use computational approaches to model, for the first time, the 3D structure of the full-length Piezo1 in an asymmetric membrane. A number of novel insights emerge: (i) Piezo1 creates a dome in the membrane with a trilobed topology that extends beyond the radius of the protein, (ii) Piezo1 changes the lipid environment in its vicinity via specific interactions with cholesterol and PIP2 molecules, (iii) changes in cholesterol concentration that change the membrane stiffness result in changes in the depth of the dome created by Piezo1, and iv) modelling of the N-terminal region that is missing from current structures modifies Piezo1 membrane footprint, suggesting the importance of this region in Piezo1 function.

biophysics↗

bioRxiv2017

Structural lipids enable the formation of functional oligomers of the eukaryotic purine symporter UapA

The role of membrane lipids in modulating eukaryotic transporter structure and function remains poorly understood. We used native mass spectrometry in combination with molecular dynamics simulations and in vivo analyses to investigate the roles of membrane lipids in the structure and transport activity of the purine transporter, UapA, from Aspergillus nidulans. We revealed that UapA exists mainly as a dimer and that two lipid molecules bind per UapA dimer. We identified three classes of phospholipids: phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) which co-purified with UapA. Delipidation of UapA caused dissociation of the dimer into individual protomers. Subsequent addition of PI or PE rescued the UapA dimer and allowed recovery of bound lipids, suggesting a central role of these lipids in stabilising the dimer. We predicted a putative lipid-binding site near the UapA dimer interface. Mutational analyses established that lipid binding at this site is essential for formation of functional UapA dimers. Our findings reveal unprecedented level of detail into the nature of UapA-lipid interactions and provide a framework for studying similar eukaryotic systems.

biophysics↗