Interdomain Conformational Changes Research Articles

Computational Method of Molecular Dynamics Simulation Identifies Insulin Receptor Binding Site 2 as the Primary Site for Insulin Binding

Background: Insulin receptor (IR) is a homo-dimeric, extensively glycosylated, disulfide-linked, transmembrane tyrosine kinase receptor. IR has two distinctive insulin binding sites, suggesting a process of sequential insulin interactions characterized by negative cooperativity. The crystal structure of the dimeric IR ectodomain [PDB: 4ZXB] provides structural bases for this theory. Objective: Identifies Insulin Receptor Binding Site 2 as the Primary Site for Insulin Binding Methods: Molecular dynamics (MD) simulation is performed to study the initial association of insulin with its receptor, leading to full interaction. MD simulation serves as a bridge between theory and experiments, enabling simulations not feasible in the lab. The study utilized the crystal structures of the insulin receptor (PDB: 4ZXB) and insulin molecule (PDB: 1MSO). GROMACS, a tool developed by Groningen University, is used for the molecular dynamics application. Prior to simulation, the receptor was prepared by restoring missing residues and removing those used for crystallization. GROMACS, compatible with several other tools, supported the MD simulations. Results: During 230 ns of all-atom, explicit-water MD simulations (0.75 million atoms), insulin and ECD-IR exhibited significant asymmetric interdomain and intersubunit conformational fluctuations without altering quaternary structures. Variations in insulin orientation and location, alongside subtle changes in residual contact of ECD-IR, coincided with these fluctuations. The insulin-IR site 2 interaction also induced interdomain conformational changes between the monomers at the subdomains L1-FnIII-2’ (L1’-FnIII-2), with initial separation studied through RMSD calculations, showing a value of 3.3Å starting at 60 ns of simulation. This protein unfolding suggests a step towards major conformational changes in ECD-IR. Conclusion: Molecular dynamics simulations provided insights into the sequential binding process and structural dynamics of the insulin-IR complex.

Protein kinase C showcases allosteric control: activation of LRRK1.

Allosteric regulation of multi-domain protein kinases provides a common mechanism to acutely control kinase activity. Protein kinase C serves as a paradigm for multi-domain proteins whose activity is exquisitely tuned by interdomain conformational changes that keep the enzyme off in the absence of appropriate stimuli, but unleash activity in response to second messenger binding. Allosteric regulation of protein kinase C signaling has been optimized not just for itself: Alessi and colleagues discover that protein kinase C phosphorylates LRRK1, a kinase with even more domains, at sites on its CORB GTPase domain to allosterically activate LRRK1.

Revealing nucleotide-induced domain movements of GTP-bound Rbg1 with long-timescale MD simulations.

Developmentally regulated GTP-binding proteins (Drgs) are highly conserved among eukaryotes and archaea and play a crucial role in such fundamental pathways as translation, differentiation, and proliferation. Ribosome-binding GTPase 1 (Rbg1), a structurally known homolog of Drgs in yeast, consists of an N-terminal HTH domain, an S5D2L domain, a typical G-domain with five G-motifs (G1-G5) responsible for binding to nucleotides, and a C-terminal TGS domain. Like other GTPases, Rbg1 is expected to undergo significant inter-domain conformational changes upon associating with nucleotides, which are transduced as cellular signals to the downstream effectors. However, nucleotide-bound structures of Rbg1 have not been resolved yet, hence the putative coupling between domain movements and the switching function of the protein remain elusive. To capture these conformational changes at an atomic level, we modeled binding of nucleotides to the protein and performed molecular dynamics simulations of GTP-bound, GDP-bound, and apo Rbg1 states for 5 µs, in 5 independent replicas for each state. We observed a significant increase in the movement of the HTH-S5D2L domains only in GTP-bound Rbg1. Close examination of trajectories shows that in GTP-bound Rbg1, the HTH-S5D2L domains move away from the G-domain resulting in a loose form of Rbg1. The angle between helix α2 of the HTH domain and helix α5 of the G-domain, representing the relative orientation of the HTH-S5D2L domains and the G-domain, exhibits a wider range of fluctuation from 50 to 122 degrees in the GTP-bound Rbg1 compared to the other systems. The above results provide valuable insights into the large-scale conformational changes of Rbg1 induced by nucleotide binding, which potentially offer a deeper understanding of the steps involved in activation of Rbg1.

Molecular mechanism of quorum sensing inhibition in Streptococcus by the phage protein paratox

Streptococcus pyogenes, or Group A Streptococcus, is a Gram-positive bacterium that can be both a human commensal and a pathogen. Central to this dichotomy are temperate bacteriophages that incorporate into the bacterial genome as prophages. These genetic elements encode both the phage proteins and the toxins harmful to the human host. One such conserved phage protein, paratox (Prx), is always found encoded adjacent to the toxin genes, and this linkage is preserved during all stages of the phage life cycle. Within S. pyogenes, Prx functions to inhibit the quorum-sensing receptor-signal pair ComRS, the master regulator of natural competence, or the ability to uptake endogenous DNA. However, the mechanism by which Prx directly binds and inhibits the receptor ComR is unknown. To understand how Prx inhibits ComR at the molecular level, we pursued an X-ray crystal structure of Prx bound to ComR. The structural data supported by solution X-ray scattering data demonstrate that Prx induces a conformational change in ComR to directly access its DNA-binding domain. Furthermore, electromobility shift assays and competition binding assays reveal that Prx effectively uncouples the interdomain conformational change required for activation of ComR via the signaling molecule XIP. Although to our knowledge the molecular mechanism of quorum-sensing inhibition by Prx is unique, it is analogous to the mechanism employed by the phage protein Aqs1 in Pseudomonas aeruginosa. Together, this demonstrates an example of convergent evolution between Gram-positive and Gram-negative phages to inhibit quorum-sensing and highlights the versatility of small phage proteins.

ATPase and Protease Domain Movements in the Bacterial AAA+ Protease FtsH Are Driven by Thermal Fluctuations

AAA+ proteases are essential players in cellular pathways of protein degradation. Elucidating their conformational behavior is key for understanding their reaction mechanism and, importantly, for elaborating our understanding of mutation-induced protease deficiencies. Here, we study the structural dynamics of the Thermotoga maritima AAA+ hexameric ring metalloprotease FtsH (TmFtsH). Using a single-molecule Förster resonance energy transfer approach to monitor ATPase and protease inter-domain conformational changes in real time, we show that TmFtsH—even in the absence of nucleotide—is a highly dynamic protease undergoing sequential transitions between five states on the second timescale. Addition of ATP does not influence the number of states or change the timescale of domain motions but affects the state occupancy distribution leading to an inter-domain compaction. These findings suggest that thermal energy, but not chemical energy, provides the major driving force for conformational switching, while ATP, through a state reequilibration, introduces directionality into this process. The TmFtsH A359V mutation, a homolog of the human pathogenic A510V mutation of paraplegin (SPG7) causing hereditary spastic paraplegia, does not affect the dynamic behavior of the protease but impairs the ATP-coupled domain compaction and, thus, may account for protease malfunctioning and pathogenesis in hereditary spastic paraplegia.

Synergistic stabilisation of NOsGC by cinaciguat and non-hydrolysable nucleotides: Evidence for sGC activator-induced communication between the heme-binding and catalytic domains

Nitric oxide sensitive guanylyl cyclase (NOsGC) is a heterodimeric enzyme consisting of one α and one β subunit. Each subunit consists of four domains: the N-terminal heme-nitric oxide oxygen binding (HNOX) domain, a PAS domain, a coiled-coil domain and the C-terminal catalytic domain. Upon activation by the endogenous ligand NO or activating drugs, NOsGC catalyses the conversion of GTP to cGMP. Although several crystal structures of the isolated domains are known, the structure of the full-length enzyme and the interdomain conformational changes during activation remain unsolved to date. In the current study, we performed protein thermal shift assays of purified NOsGC to identify discrete conformational states amenable to further analysis e.g. by crystallisation. A non-hydrolysable substrate analogue binding to the catalytic domain led to a subtle change in melting temperature. An activator drug binding to the HNOX domain led to a small increase. However, the combination of substrate analogue and activator drug led to a marked synergistic increase from 51 °C to 60 °C. This suggests reciprocal communication between HNOX domain and catalytic domain and formation of a stable activated conformation amenable to further biophysical characterization.

Mechanistic insights into staphylopine-mediated metal acquisition

Metal acquisition is vital to pathogens for successful infection within hosts. Staphylopine (StP), a broad-spectrum metallophore biosynthesized by the major human pathogen, Staphylococcus aureus, plays a central role in transition-metal acquisition and bacterial virulence. The StP-like biosynthesis loci are present in various pathogens, and the proteins responsible for StP/metal transportation have been determined. However, the molecular mechanisms of how StP/metal complexes are recognized and transported remain unknown. We report multiple structures of the extracytoplasmic solute-binding protein CntA from the StP/metal transportation system in apo form and in complex with StP and three different metals. We elucidated a sophisticated metal-bound StP recognition mechanism and determined that StP/metal binding triggers a notable interdomain conformational change in CntA. Furthermore, CRISPR/Cas9-mediated single-base substitution mutations and biochemical analysis highlight the importance of StP/metal recognition for StP/metal acquisition. These discoveries provide critical insights into the study of novel metal-acquisition mechanisms in microbes.

Interdomain Conformational Changes in Visual Arrestin upon Binding to Different Forms of Rhodopsin

Interdomain Conformational Changes in Visual Arrestin upon Binding to Different Forms of Rhodopsin

Membrane-Induced Structural Rearrangement and Identification of a Novel Membrane Anchor in Talin F2F3

Experimental challenges associated with characterization of the membrane-bound form of talin have prevented us from understanding the molecular mechanism of its membrane-dependent integrin activation. Here, utilizing what we believe to be a novel membrane mimetic model, we present a reproducible model of membrane-bound talin observed across multiple independent simulations. We characterize both local and global membrane-induced structural transitions that successfully reconcile discrepancies between biochemical and structural studies and provide insight into how talin might modulate integrin function. Membrane binding of talin, captured in unbiased simulations, proceeds through three distinct steps: initial electrostatic recruitment of the F2 subdomain to anionic lipids via several basic residues; insertion of an initially buried, conserved hydrophobic anchor into the membrane; and association of the F3 subdomain with the membrane surface through a large, interdomain conformational change. These latter two steps, to our knowledge, have not been observed or described previously. Electrostatic analysis shows talin F2F3 to be highly polarized, with a highly positive underside, which we attribute to the initial electrostatic recruitment, and a negative top face, which can help orient the protein optimally with respect to the membrane, thereby reducing the number of unproductive membrane collision events.

Localized Frustration and Binding-Induced Conformational Change in Recognition of 5S RNA by TFIIIA Zinc Finger

Protein TFIIIA is composed of nine tandemly arranged Cys2His2 zinc fingers. It can bind either to the 5S RNA gene as a transcription factor or to the 5S RNA transcript as a chaperone. Although structural and biochemical data provided valuable information on the recognition between the TFIIIIA and the 5S DNA/RNA, the involved conformational motions and energetic factors contributing to the binding affinity and specificity remain unclear. In this work, we conducted MD simulations and MM/GBSA calculations to investigate the binding-induced conformational changes in the recognition of the 5S RNA by the central three zinc fingers of TFIIIA and the energetic factors that influence the binding affinity and specificity at an atomistic level. Our results revealed drastic interdomain conformational changes between these three zinc fingers, involving the exposure/burial of several crucial DNA/RNA binding residues, which can be related to the competition between DNA and RNA for the binding of TFIIIA. We also showed that the specific recognition between finger 4/finger 6 and the 5S RNA introduces frustrations to the nonspecific interactions between finger 5 and the 5S RNA, which may be important to achieve optimal binding affinity and specificity.

SPECIFIC BINDING OF A SHORT miRNA SEQUENCE BY ZINC KNUCKLES OF Lin28: A MOLECULAR DYNAMICS SIMULATION STUDY

Protein Lin28 recognizes pre-let-7 microRNAs (miRNAs) through direct interactions between its zinc-knuckle type zinc-finger (ZnF) domains and the terminal loop of pre-let-7, resulting in the inhibition of the synthesis of mature let-7 miRNAs. Despite the physiological importance, the involved conformational changes and energetic factors contributing to the binding affinity and specificity remain unclear. We conducted molecular dynamics (MD) simulations in conjunction with molecular mechanics/generalized born surface area (MM/GBSA) and energy decomposition calculations to investigate the RNA binding-induced conformational changes of the ZnFs and the residual level energetic factors that influence the binding affinity and specificity. We showed that the binding of the RNA results in the inter-domain conformational changes of the two ZnF domains, including changes of the spatial relationships of several nucleobase-binding amino acids. We also observed mutation-induced weakening of the affinity of the Lin28–pre-let-7 binding, which reveals the importance of the stacking interactions between the side-chains of Tyr140, His148, His162 and the bases of nucleic acid G2 and G5 in the specific recognition of pre-let-7 by the ZnFs of Lin28.

Determining Interdomain Structure and Dynamics of a Retroviral Capsid Protein in the Presence of Oligomerization: Implication for Structural Transition in Capsid Assembly

Capsid (CA) proteins from all retroviruses, including HIV-1, are structurally homologous dual-domain helical proteins. They form a capsid lattice composed of unitary symmetric CA hexamers. X-ray crystallography has shown that within each hexamer a monomeric CA adopts a single conformation, where most helices are parallel to the symmetry axis. In solution, large differences in averaged NMR spin relaxation rates for the two domains were observed, suggesting they are dynamically independent. One relevant question for the capsid assembly remains: whether the interdomain conformer within a hexamer unit needs to be induced or pre-exists within the conformational space of a monomeric CA. The latter seems more consistent with the relaxation data. However, possible CA protein oligomerization and the structure of each domain will affect relaxation measurements and data interpretation. This study, using CA proteins from equine infectious anemia virus (EIAV) as an example, demonstrates a linear extrapolation approach to obtain backbone (15)N spin relaxation time ratios T1/T2 for a monomeric EIAV-CA in the presence of oligomerization equilibrium. The interdomain motion turns out to be limited. The large difference in the domain averaged <T1/T2> for a CA monomer is a consequence of the orthogonal distributions of helices in the two domains. The new monomeric interdomain conformation in solution is significantly different from that in CA hexamer. Therefore, if capsid assembly follows a nucleation-propagation process, the interdomain conformational change might be a key step during the nucleation, as the configuration in hexagonal assembly is never formed by diffusion of its two domains in solution.

Crystal Structure of the Filamin N-Terminal Region Reveals a Hinge between the Actin Binding and First Repeat Domains

The filamin proteins cross-link F-actin and interact with protein partners to integrate both extracellular and intracellular signalling events with the cytoskeleton and to provide mechanoprotection and sensing to cells. The filamins are large, flexible, multi-domain homodimers with the interactions between domains important for protein function. The crystal structure of the N-terminal region of filamin B, containing the actin binding domain (ABD) and the first filamin repeat (FR1) domain, reveals an extended two-domain conformation with no interaction between the ABD and FR1 other than the connecting linker region. The two FLNB347 structures in the crystallographic asymmetric unit exhibit differing relative domain orientations providing the first high-resolution structural characterisation of a filamin inter-domain conformational change. The structure reveals a new hinge in the linker region between ABD and FR1 that is ideally positioned to orient the ABD for actin binding and adds to the previously described hinge regions, hinge 1 (between repeats 15 and 16) and hinge 2 (repeats 23 and 24), providing an additional mechanism by which filamin can exhibit inter-domain flexibility. The extended structure, with the absence of interactions between the domains, implies that any conformational rearrangements required for actin binding by the ABD, as observed for homologous proteins, can freely occur without being influenced by FR1. The ABD retains its previously observed compact conformation. FR1 exhibits a filamin immunoglobulin-like domain fold with a closed C-D β-strand groove, in contrast to filamin repeats that bind protein partners with this region of the domain surface.

Enhancement of the Structural Stability of Full-Length Clostridial Collagenase by Calcium Ions

The clostridial collagenases G and H are multidomain proteins. For collagen digestion, the domain arrangement is likely to play an important role in collagen binding and hydrolysis. In this study, the full-length collagenase H protein from Clostridium histolyticum was expressed in Escherichia coli and purified. The N-terminal amino acid of the purified protein was Ala31. The expressed protein showed enzymatic activity against azocoll as a substrate. To investigate the role of Ca(2+) in providing structural stability to the full-length collagenase H, biophysical measurements were conducted using the recombinant protein. Size exclusion chromatography revealed that the Ca(2+) chelation by EGTA induced interdomain conformational changes. Dynamic light scattering measurements showed an increase in the percent polydispersity as the Ca(2+) was chelated, suggesting an increase in protein flexibility. In addition to these conformational changes, differential scanning fluorimetry measurements revealed that the thermostability was decreased by Ca(2+) chelation, in comparison with the thermal melting point (T(m)). The melting point changed from 54 to 49°C by the Ca(2+) chelation, and it was restored to 54°C by the addition of excess Ca(2+). These results indicated that the interdomain flexibility and the domain arrangement of full-length collagenase H are reversibly regulated by Ca(2+).

A Coevolutionary Residue Network at the Site of a Functionally Important Conformational Change in a Phosphohexomutase Enzyme Family

Coevolution analyses identify residues that co-vary with each other during evolution, revealing sequence relationships unobservable from traditional multiple sequence alignments. Here we describe a coevolutionary analysis of phosphomannomutase/phosphoglucomutase (PMM/PGM), a widespread and diverse enzyme family involved in carbohydrate biosynthesis. Mutual information and graph theory were utilized to identify a network of highly connected residues with high significance. An examination of the most tightly connected regions of the coevolutionary network reveals that most of the involved residues are localized near an interdomain interface of this enzyme, known to be the site of a functionally important conformational change. The roles of four interface residues found in this network were examined via site-directed mutagenesis and kinetic characterization. For three of these residues, mutation to alanine reduces enzyme specificity to ∼10% or less of wild-type, while the other has ∼45% activity of wild-type enzyme. An additional mutant of an interface residue that is not densely connected in the coevolutionary network was also characterized, and shows no change in activity relative to wild-type enzyme. The results of these studies are interpreted in the context of structural and functional data on PMM/PGM. Together, they demonstrate that a network of coevolving residues links the highly conserved active site with the interdomain conformational change necessary for the multi-step catalytic reaction. This work adds to our understanding of the functional roles of coevolving residue networks, and has implications for the definition of catalytically important residues.

Conformational Analysis Reveals Distinct Mechanical Transduction Mechanisms of RecA-Like Molecular Motors

A majority of ATP-dependent molecular motors are RecA-like proteins, performing diverse functions in biology. These RecA-like molecular motors consist of a highly conserved core containing the ATP-binding site. Here I examined how ATP binding within this core is coupled to the conformational changes of different RecA-like molecular motors. This study showed that dense hydrogen bond networks are prevalent in the ATP binding sites of RecA-like molecular motors when ATP is present, but the conformations of different RecA-like motors in the apo state are varying. Conserved hydrogen bond networks and conformational changes revealed two major mechanical transduction mechanisms [1]: (1) intra-domain conformational changes and (2) inter-domain conformational changes. The intra-domain mechanism has a significant hydrogen bond rearrangement within the domain containing the P-loop, causing relative motion between two parts of the protein. The inter-domain mechanism exhibits little conformational change in the P-loop domain. Instead, the major conformational change is observed between the P-loop domain and an adjacent domain or subunit containing the arginine finger. These differences in the mechanical transduction mechanisms may link to the underlying energy surface governing a Brownian ratchet or a power stroke. This work is supported by NSF grant CMMI-1125760. [1] Liao, J.-C., (2011) Mechanical Transduction Mechanisms of RecA-like Molecular Motors, J Biomol Struct Dyn, in press.

Capturing Spontaneous Membrane Insertion and Membrane-Induced Conformational Changes of Talin at an Atomic Resolution

Integrins are a diverse set of proteins that play a central role in complex biological processes, such as tumor metastasis and thrombus formation. The integrin heterodimer is often expressed in a low-affinity, inactive state, relying on specific cytoplasmic or extracellular signals for its activation. The cytoskeletal-associated protein talin constitutes one of the major activation pathways of integrin through a membrane-mediated mechanism. While the involvement of activated, membrane-bound talin in this process is well established, atomic details of membrane binding of talin and of talin-dependent integrin activation have been lacking. Using our novel, highly mobile membrane mimetic simulation system, we have successfully captured complete insertion of the talin head domain (THD) in a phosphatidylserine membrane in three independent unbiased simulations, revealing key molecular events involved in the process. The THD is initially recruited to the membrane via the documented membrane orientation patch (MOP), consisting of a large number of positively charged residues. Electrostatic potential calculations revealed THD to be highly polarized, providing a potential mechanism explaining how the protein is aligns for optimal encounter with the membrane. We also observe a large, membrane-induced interdomain conformational change (>2.5 nm), which brings the F3 subdomain into contact with the anionic membrane via residues K325, N326, and K327. This result explains how F2 and F3 subdomains can simultaneously bind the membrane, a biochemically established aspect that could not be explained by the crystal structure. Moreover, we characterize a phenylalanine-rich region as the hydrophobic membrane anchor, consisting mainly of F261 and F283, which is released through the snorkeling motion of a few critical lysine residues within the membrane. Although such an anchor has been hypothesized to exist, none had been identified prior to this study.

Effects of Ethanol on Conformational Changes of Akt Studied by Chemical Cross-Linking, Mass Spectrometry, and 18O Labeling

Although PI3K/Akt signaling that regulates neuronal survival has been implicated in the deleterious effects of ethanol on the central nervous system, underlying molecular mechanisms have not been fully elucidated. Akt-membrane interaction is a prerequisite step for Akt activation since it induces interdomain conformational changes to an open conformer that allows Akt phosphorylation by upstream kinases. In this study, we investigated the effect of ethanol on Akt activation by quantitatively probing Akt conformation using chemical cross-linking, (18)O labeling and mass spectrometry. We found that ethanol at pharmacologically relevant concentrations (20 or 170 mM) directly interacts with Akt and alters the local pleckstrin homology domain configuration near the PIP(3)-binding site. We also found that ethanol significantly impairs subsequent membrane-induced interdomain conformational changes needed for Akt activation. The observed alteration of Akt conformation caused by ethanol during the activation sequence provides a new molecular basis for the effects of ethanol on Akt signaling. The in vitro conformation-based approach employed in this study should also be useful in probing the molecular mechanisms for the action of ethanol or drugs on other signaling proteins, particularly for those undergoing dramatic conformational change during activation processes such as members of AGC kinase super family.

Mechanical Transduction Mechanisms of RecA-Like Molecular Motors

A majority of ATP-dependent molecular motors are RecA-like proteins, performing diverse functions in biology. These RecA-like molecular motors consist of a highly conserved core containing the ATP-binding site. Here I examined how ATP binding within this core is coupled to the conformational changes of different RecA-like molecular motors. Conserved hydrogen bond networks and conformational changes revealed two major mechanical transduction mechanisms: (1) intra-domain conformational changes and (2) inter-domain conformational changes. The intra-domain mechanism has a significant hydrogen bond rearrangement within the domain containing the P-loop, causing relative motion between two parts of the protein. The inter-domain mechanism exhibits little conformational change in the P-loop domain. Instead, the major conformational change is observed between the P-loop domain and an adjacent domain or subunit containing the arginine finger. These differences in the mechanical transduction mechanisms may link to the underlying energy surface governing a Brownian ratchet or a power stroke.

Dynamic Characterization of the Membrane-Mediated Activation of Integrin by Talin At Atomic Reoslution

Abstract 2191Integrins are a diverse set of proteins that play a central role in many complex biological processes, such as embryonic development, tumor metastasis, and thrombus formation. The integrin heterodimer is often expressed in a low-affinity, inactive state, relying on specific cytoplasmic or extracellular signals for its activation. One of the major activation pathways that has come to the forefront of integrin research is the membrane-mediated activation of integrin by the cytoskeletal-associated protein talin. While the interaction between talin and integrin is well-established, an atomic-resolution description of the membrane-binding process of talin and of talin-dependent integrin activation has been lacking.Here, we present a study describing the membrane insertion process of the talin head domain (THD) and its subsequent interaction with the transmembrane domain of integrin αIIbβ3. Using our novel, highly mobile membrane mimetic simulation system, we simulated complete membrane insertion of THD in a phosphatidylserine (PS) membrane a total of six (6) times, revealing key molecular events involved in the process. The THD is initially recruited to the membrane via the documented membrane orientation patch (MOP), consisting of a large number of positively charged residues. However, we also observe a large, interdomain conformational change (> 2.5 nm), which brings the F3 subdomain into contact with the surface of the anionic membrane via residues K325, N326, and K327. Moreover, we characterize a novel, phenylalanine-rich region as the hydrophobic membrane anchor, consisting mainly of F261 and F283, which is released through the snorkeling motion of a few critical lysine residues within the membrane. Although such an anchor has been hypothesized to exist, none had been identified prior to this study.Using the membrane-bound model of the THD generated in these simulations, we have also simulated the interaction between talin and the integrin β3 transmembrane domain. We were able to identify the well-known NPxY-PTB binding motif, in addition to a non-specific intermolecular hydrophobic pocket, which helps talin bind to the integrin β3 tail. Once bound to integrin β3, talin induces a bend of over 30° in the transmembrane domain at the surface of the membrane (Figure 1), which causes the burial of the conserved D723 within the lipid head-group region; this phenomenon has been observed in all cases. We believe this is a mechanism for integrin activation whereby the conserved salt bridge is mechanically broken by the THD, leading to separation of the α- and β-helices. [Display omitted] Finally, the separation of the the α- and β-tails apart wasstudied using slow pulling simulations. These studies have shown that the cytoplasmic ends of the helices interact rather loosely, while the extracellular endsare held together via tight packing of the helices. Free energy profiles of mutants in this region have shown significant decreases in stability of the “off-state”, while mutants in the cytoplasmic end do not show as significant an effect on the free energy landscape of the dimer. Disclosures:No relevant conflicts of interest to declare.