Allosteric inhibition of the epidermal growth factor receptor through disruption of transmembrane interactions.

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase (RTK) commonly targeted for inhibition by anticancer therapeutics. Current therapeutics target EGFR's kinase domain or extracellular region. However, these types of inhibitors are not specific for tumors over healthy tissue and therefore cause undesirable side effects. Our lab has recently developed a new strategy to regulate RTK activity by designing a peptide that specifically binds to the transmembrane (TM) region of the RTK to allosterically modify kinase activity. These peptides are acidity-responsive, allowing them to preferentially target acidic environments like tumors. We have applied this strategy to EGFR and created the PET1 peptide. We observed that PET1 behaves as a pH-responsive peptide that modulates the configuration of the EGFR TM through a direct interaction. Our data indicated that PET1 inhibits EGFR-mediated cell migration. Finally, we investigated the mechanism of inhibition through molecular dynamics simulations, which showed that PET1 sits between the two EGFR TM helices; this molecular mechanism was additionally supported by AlphaFold-Multimer predictions. We propose that the PET1-induced disruption of native TM interactions disturbs the conformation of the kinase domain in such a way that it inhibits EGFR's ability to send migratory cell signals. This study is a proof-of-concept that acidity-responsive membrane peptide ligands can be generally applied to RTKs. In addition, PET1 constitutes a viable approach to therapeutically target the TM of EGFR.

Raf promotes dimerization of the Ras G-domain with increased allosteric connections.

Ras dimerization is critical for Raf activation. Here we show that the Ras binding domain of Raf (Raf-RBD) induces robust Ras dimerization at low surface densities on supported lipid bilayers and, to a lesser extent, in solution as observed by size exclusion chromatography and confirmed by SAXS. Community network analysis based on molecular dynamics simulations shows robust allosteric connections linking the two Raf-RBD D113 residues located in the Galectin scaffold protein binding site of each Raf-RBD molecule and 85 Å apart on opposite ends of the dimer complex. Our results suggest that Raf-RBD binding and Ras dimerization are concerted events that lead to a high-affinity signaling complex at the membrane that we propose is an essential unit in the macromolecular assembly of higher order Ras/Raf/Galectin complexes important for signaling through the Ras/Raf/MEK/ERK pathway.

NMR identification of a conserved Drp1 cardiolipin-binding motif essential for stress-induced mitochondrial fission.

Mitochondria form tubular networks that undergo coordinated cycles of fission and fusion. Emerging evidence suggests that a direct yet unresolved interaction of the mechanoenzymatic GTPase dynamin-related protein 1 (Drp1) with mitochondrial outer membrane-localized cardiolipin (CL), externalized under stress conditions including mitophagy, catalyzes essential mitochondrial hyperfragmentation. Here, using a comprehensive set of structural, biophysical, and cell biological tools, we have uncovered a CL-binding motif (CBM) conserved between the Drp1 variable domain (VD) and the unrelated ADP/ATP carrier (AAC/ANT) that intercalates into the membrane core to effect specific CL interactions. CBM mutations that weaken VD-CL interactions manifestly impair Drp1-dependent fission under stress conditions and induce "donut" mitochondria formation. Importantly, VD membrane insertion and GTP-dependent conformational rearrangements mediate only transient CL nonbilayer topological forays and high local membrane constriction, indicating that Drp1-CL interactions alone are insufficient for fission. Our studies establish the structural and mechanistic bases of Drp1-CL interactions in stress-induced mitochondrial fission.

A proteome-scale analysis of vertebrate protein amino acid occurrence: Thermoadaptation shows a correlation with protein solvation but less so with dynamics.

Despite differences in behaviors and living conditions, vertebrate organisms share the great majority of proteins, often with subtle differences in amino acid sequence. Here, we present a simple way to analyze the difference in amino acid occurrence by comparing highly homologous proteins on a subproteome level between several vertebrate model organisms. Specifically, we use this method to identify a pattern of amino acid conservation as well as a shift in amino acid occurrence between homeotherms (warm-blooded species) and poikilotherms (cold-blooded species). Importantly, this general analysis and a specific example further establish a broad correlation, if not likely connection between the thermal adaptation of protein sequences and two of their physical features: on average a change in their protein dynamics and, even more strongly, in their solvation. For poikilotherms, such as frog and fish, the lower body temperature is expected to increase the protein-protein interaction due to a decrease in protein internal dynamics. In order to counteract the tendency for enhanced binding caused by low temperatures, poikilotherms enhance the solvation of their proteins by favoring polar amino acids. This feature appears to dominate over possible changes in dynamics for some proteins. The results suggest that a general trend for amino acid choice is part of the mechanism for thermoadaptation of vertebrate organisms at the molecular level.

Letting go: Deep computational modeling insights into pH-dependent calcium affinity.

Calcium and other cofactors can feature as key additions to a molecular interface, to the extent that the cofactor is completely buried in the bound state. How can such an interaction be regulated then? The answer: By facilitating a switch through an allosteric network. Although a number of unbinding mechanisms are being characterized, an extensive computational study by Joswig et al. reveals a detailed model for the pattern recognition receptor langerin.

Interactions between semaphorins and plexin-neuropilin receptor complexes in the membranes of live cells.

Signaling of semaphorin ligands via their plexin-neuropilin receptors is involved in tissue patterning in the developing embryo. These proteins play roles in cell migration and adhesion but are also important in disease etiology, including in cancer angiogenesis and metastasis. While some structures of the soluble domains of these receptors have been determined, the conformations of the full-length receptor complexes are just beginning to be elucidated, especially within the context of the plasma membrane. Pulsed-interleaved excitation fluorescence cross-correlation spectroscopy allows direct insight into the formation of protein-protein interactions in the membranes of live cells. Here, we investigated the homodimerization of neuropilin-1 (Nrp1), plexin A2, plexin A4, and plexin D1 using pulsed-interleaved excitation fluorescence cross-correlation spectroscopy. Consistent with previous studies, we found that Nrp1, plexin A2, and plexin A4 are present as dimers in the absence of exogenous ligand. Plexin D1, on the other hand, was monomeric under similar conditions, which had not been previously reported. We also found that plexin A2 and A4 assemble into a heteromeric complex. Stimulation with semaphorin 3A or semaphorin 3C neither disrupts nor enhances the dimerization of the receptors when expressed alone, suggesting that activation involves a conformational change rather than a shift in the monomer-dimer equilibrium. However, upon stimulation with semaphorin 3C, plexin D1 and Nrp1 form a heteromeric complex. This analysis of interactions provides a complementary approach to the existing structural and biochemical data that will aid in the development of new therapeutic strategies to target these receptors in cancer.

Plexin-Bs enhance their GAP activity with a novel activation switch loop generating a cooperative enzyme.

Plexins receive guidance cues from semaphorin ligands and transmit their signal through the plasma membrane. This family of proteins is unique amongst single-pass transmembrane receptors as their intracellular regions interact directly with several small GTPases, which regulate cytoskeletal dynamics and cell adhesion. Here, we characterize the GTPase Activating Protein (GAP) function of Plexin-B1 and find that a cooperative GAP activity towards the substrate GTPase, Rap1b, is associated with the N-terminal Juxtamembrane region of Plexin-B1. Importantly, we unveil an activation mechanism of Plexin-B1 by identifying a novel functional loop which partially blocks Rap1b entry into the plexin GAP domain. Consistent with the concept of allokairy developed for other systems, Plexin-B activity is increased by an apparent substrate-mediated cooperative effect. Simulations and mutagenesis suggest the repositioned JM conformation is stabilized by the new activation switch loop when the active site is occupied, giving rise to faster enzymatic turnover and cooperative behavior. The biological implications, essentially those of a threshold behavior for cell migration, are discussed.

Neuropilin-1 assists SARS-CoV-2 infection by stimulating the separation of Spike protein S1 and S2.

The cell surface receptor Neuropilin-1 (Nrp1) was recently identified as a host factor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry. The Spike protein of SARS-CoV-2 is cleaved into two segments, the S1 (residues (res.) 1-685) and the S2 (res. 686-1273) domains by furin protease. Nrp1 predominantly binds to the C-terminal RRAR amino acid motif (res. 682-685) of the S1 domain. In this study, we firstly modeled the association of an Nrp1 protein (consisting of domains a2-b1-b2) with the Spike protein. Next, we studied the separation of S2 from the S1 domain, with and without Nrp1 bound, by utilizing molecular dynamics pulling simulations. During the separation, Nrp1 stabilizes the S1 C-terminal region (res. 640-685) and thereby assists the detachment of S2 N-terminal region (res. 686-700). Without Nrp1 bound, S1 tends to become stretched, whereas the bound Nrp1 stimulates an earlier separation of S2 from the S1 domain. The liberated S2 domain is known to mediate the fusion of virus and host membranes; thus, Nrp1 likely increases virus infectivity by facilitating the S1 and S2 separation. We further analyzed the possible topological structure of the SARS-CoV-2 Spike protein when bound with Nrp1 and angiotensin-converting enzyme 2 (ACE2). Understanding of such an Nrp1-assisted viral infection opens the gate for the generation of protein-protein inhibitors, such as antibodies, which could attenuate the infection mechanism and protect certain cells in a future Nrp1-ACE2 targeted combination therapy.

Structural and Functional Insights into the Transmembrane Domain Association of Eph Receptors.

Eph receptors are the largest family of receptor tyrosine kinases and by interactions with ephrin ligands mediate a myriad of processes from embryonic development to adult tissue homeostasis. The interaction of Eph receptors, especially at their transmembrane (TM) domains is key to understanding their mechanism of signal transduction across cellular membranes. We review the structural and functional aspects of EphA1/A2 association and the techniques used to investigate their TM domains: NMR, molecular modelling/dynamics simulations and fluorescence. We also introduce transmembrane peptides, which can be used to alter Eph receptor signaling and we provide a perspective for future studies.

K-Ras G-domain binding with signaling lipid phosphatidylinositol (4,5)-phosphate (PIP2): membrane association, protein orientation, and function.

Ras genes potently drive human cancers, with mutated proto-oncogene GTPase KRAS4B (K-Ras4B) being the most abundant isoform. Targeted inhibition of oncogenic gene products is considered the "holy grail" of present-day cancer therapy, and recent discoveries of small-molecule KRas4B inhibitors were made thanks to a deeper understanding of the structure and dynamics of this GTPase. Because interactions with biological membranes are key for Ras function, Ras-lipid interactions have become a major focus, especially because such interactions evidently involve both the Ras C terminus for lipid anchoring and its G-protein domain. Here, using NMR spectroscopy and molecular dynamics simulations complemented by biophysical- and cell-biology assays, we investigated the interaction between K-Ras4B with the signaling lipid phosphatidylinositol (4,5)-phosphate (PIP2). We discovered that the ß2 and ß3 strands as well as helices 4 and 5 of the GTPase G-domain bind to PIP2 and identified the specific residues in these structural elements employed in these interactions, likely occurring in two K-Ras4B orientation states relative to the membrane. Importantly, we found that some of these residues known to be oncogenic when mutated (D47K, D92N, K104M, and D126N) are critical for K-Ras-mediated transformation of fibroblast cells, but do not substantially affect basal and assisted nucleotide hydrolysis and exchange. Moreover, the K104M substitution abolished localization of K-Ras to the plasma membrane. The findings suggest that specific G-domain residues can critically regulate Ras function by mediating interactions with membrane-associated PIP2 lipids; these insights that may inform the future design of therapeutic reagents targeting Ras activity.

Computational Design of Myristoylated Cell-Penetrating Peptides Targeting Oncogenic K-Ras.G12D at the Effector-Binding Membrane Interface.

A number of small inhibitors have been developed in recent years to target the cancer-driving protein, K-Ras. In this study, we propose and design a novel way of targeting oncogenic K-Ras4B.G12D with myristoylated cell-penetrating peptides which become membrane-anchored and lock the protein into an inactive state. In all atom molecular dynamics simulations, such peptides associate with K-Ras4B exclusively at the effector-binding region, which, in turn, is expected to hinder the binding of downstream effector proteins (e.g., C-Raf). The myristoylated R9 (Arg9) peptide locks K-Ras4B.G12D into orientations that are unfavorable for effector binding. After breaking the cyclic structure and myristoylation, a cell-penetrating peptide cyclorasin 9A5, which was designed for targeting the Ras/Raf interface, is also found to be effective in targeting the Ras/membrane interface. The myristoylated peptides likely have high cell permeability because of their mixed cationic/hydrophobic character at the N-terminus, while simultaneously the subsequent multiple charges help to maintain a close association of the peptide with the K-Ras4B.G12D effector-binding lobe. Targeting protein-membrane interfaces is starting to attract attention very recently, thanks to our understanding of the signaling mechanism of an increased number of peripheral membrane proteins. The strategy used in this study has potential applications in the design of drugs against K-Ras4B-driven cancers. It also provides insights into the general principles of targeting protein-membrane interfaces.

Cyclase-associated protein 1 (CAP1) is a prenyl-binding partner of Rap1 GTPase.

Rap1 proteins are members of the Ras subfamily of small GTPases involved in many biological responses, including adhesion, cell proliferation, and differentiation. Like all small GTPases, they work as molecular allosteric units that are active in signaling only when associated with the proper membrane compartment. Prenylation, occurring in the cytosol, is an enzymatic posttranslational event that anchors small GTPases at the membrane, and prenyl-binding proteins are needed to mask the cytoplasm-exposed lipid during transit to the target membrane. However, several of these proteins still await discovery. In this study, we report that cyclase-associated protein 1 (CAP1) binds Rap1. We found that this binding is GTP-independent, does not involve Rap1's effector domain, and is fully contained in its C-terminal hypervariable region (HVR). Furthermore, Rap1 prenylation was required for high-affinity interactions with CAP1 in a geranylgeranyl-specific manner. The prenyl binding specifically involved CAP1's C-terminal hydrophobic ß-sheet domain. We present a combination of experimental and computational approaches, yielding a model whereby the high-affinity binding between Rap1 and CAP1 involves electrostatic and nonpolar side-chain interactions between Rap1's HVR residues, lipid, and CAP1 ß-sheet domain. The binding was stabilized by the lipid insertion into the ß-solenoid whose interior was occupied by nonpolar side chains. This model was reminiscent of the recently solved structure of the PDEδ-K-Ras complex; accordingly, disruptors of this complex, e.g. deltarasin, blocked the Rap1-CAP1 interaction. These findings indicate that CAP1 is a geranylgeranyl-binding partner of Rap1.

Dissociation of a Dynamic Protein Complex Studied by All-Atom Molecular Simulations.

The process of protein complex dissociation remains to be understood at the atomic level of detail. Computers now allow microsecond timescale molecular-dynamics simulations, which make the visualization of such processes possible. Here, we investigated the dissociation process of the EphA2-SHIP2 SAM-SAM domain heterodimer complex using unrestrained all-atom molecular-dynamics simulations. Previous studies on this system have shown that alternate configurations are sampled, that their interconversion can be fast, and that the complex is dynamic by nature. Starting from different NMR-derived structures, mutants were designed to stabilize a subset of configurations by swapping ion pairs across the protein-protein interface. We focused on two mutants, K956D/D1235K and R957D/D1223R, with attenuated binding affinity compared with the wild-type proteins. In contrast to calculations on the wild-type complexes, the majority of simulations of these mutants showed protein dissociation within 2.4 µs. During the separation process, we observed domain rotation and pivoting as well as a translation and simultaneous rolling, typically to alternate and weaker binding interfaces. Several unsuccessful recapturing attempts occurred once the domains were moderately separated. An analysis of protein solvation suggests that the dissociation process correlates with a progressive loss of protein-protein contacts. Furthermore, an evaluation of internal protein dynamics using quasi-harmonic and order parameter analyses indicates that changes in protein internal motions are expected to contribute significantly to the thermodynamics of protein dissociation. Considering protein association as the reverse of the separation process, the initial role of charged/polar interactions is emphasized, followed by changes in protein and solvent dynamics. The trajectories show that protein separation does not follow a single distinct pathway, but suggest that the mechanism of dissociation is common in that it initially involves transitions to surfaces with fewer, less favorable contacts compared with those seen in the fully formed complex.

Binding and function of phosphotyrosines of the Ephrin A2 (EphA2) receptor using synthetic sterile α motif (SAM) domains.

The sterile α motif (SAM) domain of the ephrin receptor tyrosine kinase, EphA2, undergoes tyrosine phosphorylation, but the effect of phosphorylation on the structure and interactions of the receptor is unknown. Studies to address these questions have been hindered by the difficulty of obtaining site-specifically phosphorylated proteins in adequate amounts. Here, we describe the use of chemically synthesized and specifically modified domain-length peptides to study the behavior of phosphorylated EphA2 SAM domains. We show that tyrosine phosphorylation of any of the three tyrosines, Tyr(921), Tyr(930), and Tyr(960), has a surprisingly small effect on the EphA2 SAM structure and stability. However, phosphorylation at Tyr(921) and Tyr(930) enables differential binding to the Src homology 2 domain of the adaptor protein Grb7, which we propose will lead to distinct functional outcomes. Setting up different signaling platforms defined by selective interactions with adaptor proteins thus adds another level of regulation to EphA2 signaling.

Local Structures in Proteins from Microsecond Molecular Dynamics Simulations: 2. The Role of Symmetry in GTPase Binding and Dimer Formation.

The Rho GTPase binding domain of Plexin-B1 (RBD) prevails in solution as dimer. Under appropriate circumstances, it binds the small GTPase Rac1 to yield the complex RBD-Rac1. Here, we study RBD dimerization and complex formation from a symmetry-based perspective using data derived from 1 µs long MD simulations. The quantities investigated are the local potentials, u(MD), prevailing at the N-H sites of the protein. These potentials are statistical in character providing an empirical description of the local structure. To establish more methodical description, a method for approximating them by explicit functions, u(simulated), was developed in the preceding article in this journal issue. These functions are combinations of analytical Wigner functions, DL,K, belonging to the D2h point group. The D2h subgroups Ag and B2u are found to dominate u(simulated); the B1u subgroup contributes in some cases. The Ag (B2u) functions have axial or rhombic symmetry. For the first time, local potentials in proteins can be quantitatively characterized in terms of their strength (rhombicity) evaluated by axial Ag (rhombic Ag and B2u) contributions. Until now, the chain-segment [ß3-L3-ß4] and to some extent the α2-helix have been associated with GTPase binding. Here, we find that this process causes an increase (decrease) in the potential strength of ß3 and ß4 (the preceding L2 loop and the remote chain-segment [(α2-helix)-(α2/ß5-turn)-(ß5-strand)]), suggesting effects of counterbalancing and allostery. There is evidence for the L2 loop being associated with RBD-GTPase binding. Until now only the L4 loop has been associated with RBD dimerization. The latter process is found to cause an increase (decrease) in the potential strength and rhombicity of the L4 loop (the adjacent chain-segment [(α2-helix)-(α2/ß5-turn)-(ß5-strand)]), suggesting counterbalancing activity. On average, the RBD dimer features stronger local potentials than RBD-Rac1. The novel information inherent in these findings is mesoscopic in character. Prospects of interest include exploring relation to atomistic force-field parameters.

Local Structures in Proteins from Microsecond Molecular Dynamics Simulations: A Symmetry-Based Perspective.

We report on a new method for the characterization of local structures in proteins based on extensive molecular dynamics (MD) simulations, here, 1 µs in length. The N-H bond of the Rho GTPase binding domain of plexin-B1 (RBD) serves as a probe and the potential, u(MD), which restricts its internal motion, as a qualifier of the local dynamic structure. u(MD) is derived from the MD trajectory as a function of the polar angles, (θ, φ), which specify the N-H orientation in the protein. u(MD) is statistical in character yielding empirical descriptions. To establish more insightful methodical descriptions, we develop a comprehensive method which approximates u(MD) by combinations of analytical Wigner functions that belong to the D2h point group. These combinations, called u(simulated), make it possible to gain a new perspective of local dynamic structures in proteins based on explicit potentials/free energy surfaces and associated probability densities, entropy, and ordering. A simpler method was developed previously using 100 ns MD simulations. In that case, the traditional "perpendicular N-H ordering" setting centered at Cα-Cα with (θ, φ) = (90, 90) and generally, featuring positive φ, prevailed. u(MD) derived from 1 µs MD simulations is considerably more complex requiring substantial model enhancement. The enhanced method applies to the well-structured sections of the RBD. It only applies partly to its loops where u(MD) extends into the negative-φ region where we detect nonperpendicular N-H ordering. This arrangement requires devising new reference structures and making substantial algorithmic changes, to be performed in future work. Here, we focus on developing the comprehensive method and using it to investigate perpendicular ordering settings. We find that secondary structures (loops) exhibit varying (virtually invariant) potentials with Ag, B2u, and B1u (Ag and B2u) D2h symmetry. Application to RBD dimerization and RBD binding to the GTPase Rac1 is described in the subsequent article. Applications to other probes, proteins, and biological functions, based on explicit local potentials, probability densities, entropy, and ordering, are possible.

Cholesterol inhibits assembly and activation of the EphA2 receptor.

The receptor tyrosine kinase EphA2 drives cancer malignancy by facilitating metastasis. EphA2 can be found in different self-assembly states: as a monomer, dimer, and oligomer. However, our understanding remains limited regarding which EphA2 state is responsible for driving pro-metastatic signaling. To address this limitation, we have developed SiMPull-POP, a single-molecule method for accurate quantification of membrane protein self-assembly. Our experiments revealed that a reduction of plasma membrane cholesterol strongly promoted EphA2 self-assembly. Indeed, low cholesterol caused a similar effect to the EphA2 ligand ephrinA1-Fc. These results indicate that cholesterol inhibits EphA2 assembly. Phosphorylation studies in different cell lines revealed that low cholesterol increased phospho-serine levels, the signature of oncogenic signaling. Investigation of the mechanism that cholesterol uses to inhibit the assembly and activity of EphA2 indicate an in-trans effect, where EphA2 is phosphorylated by protein kinase A downstream of beta-adrenergic receptor activity, which cholesterol also inhibits. Our study not only provides new mechanistic insights on EphA2 oncogenic function, but also suggests that cholesterol acts as a molecular safeguard mechanism that prevents uncontrolled self-assembly and activation of EphA2.

Molecular simulations of a dynamic protein complex: role of salt-bridges and polar interactions in configurational transitions.

Ion charge pairs and hydrogen bonds have been extensively studied for their roles in stabilizing protein complexes and in steering the process of protein association. Recently, it has become clear that some protein complexes are dynamic in that they interconvert between several alternate configurations. We have previously characterized one such system: the EphA2:SHIP2 SAM-SAM heterodimer by solution NMR. Here we carried out extensive all-atom molecular-dynamics simulations on a microsecond time-scale starting with different NMR-derived structures for the complex. Transitions are observed between several discernible configurations at average time intervals of 50-100 ns. The domains reorient relative to one another by substantial rotation and a slight shifting of the interfaces. Bifurcated and intermediary salt-bridge and hydrogen-bond interactions play a role in the transitions in a process that can be described as moving along a "monkey-bar". We notice an increased density of salt bridges near protein interaction surfaces that appear to enable these transitions, also suggesting why the trajectories can become kinetically hindered in regions where fewer of such interactions are possible. In this context, even microsecond molecular-dynamics simulations are not sufficient to sample the energy landscape unless the structures remain close to their experimentally derived low-energy configurations.