MHC-II dynamics are maintained in HLA-DR allotypes to ensure catalyzed peptide exchange.

Presentation of antigenic peptides by major histocompatibility complex class II (MHC-II) proteins determines T helper cell reactivity. The MHC-II genetic locus displays a large degree of allelic polymorphism influencing the peptide repertoire presented by the resulting MHC-II protein allotypes. During antigen processing, the human leukocyte antigen (HLA) molecule HLA-DM (DM) encounters these distinct allotypes and catalyzes exchange of the placeholder peptide CLIP by exploiting dynamic features of MHC-II. Here, we investigate 12 highly abundant CLIP-bound HLA-DRB1 allotypes and correlate dynamics to catalysis by DM. Despite large differences in thermodynamic stability, peptide exchange rates fall into a target range that maintains DM responsiveness. A DM-susceptible conformation is conserved in MHC-II molecules, and allosteric coupling between polymorphic sites affects dynamic states that influence DM catalysis. As exemplified for rheumatoid arthritis, we postulate that intrinsic dynamic features of peptide-MHC-II complexes contribute to the association of individual MHC-II allotypes with autoimmune disease.

PySFD: comprehensive molecular insights from significant feature differences detected among many simulated ensembles.

MOTIVATION: Many modeling analyses of molecular dynamics (MD) simulations are based on a definition of states that can be (groups of) clusters of simulation frames in a feature space composed of molecular coordinates. With increasing dimension of this feature space (due to the increasing size or complexity of a simulated molecule), it becomes very difficult to cluster the underlying MD data and estimate a statistically robust model. To mitigate this "curse of dimensionality", one can reduce the feature space, e.g., with principal component or time-lagged independent component analysis transformations, focusing the analysis on the most important modes of transitions. In practice, however, all these reduction strategies may neglect important molecular details that are susceptible to experimental verification. RESULTS: To recover such molecular details, I have developed PySFD (Significant Feature Differences analyzer for Python), a multi-processing software package that efficiently selects significantly different features of any user-defined feature type among potentially many different simulated state ensembles, such as meta-stable states of a Markov State Model (MSM). Applying PySFD on MSMs of an aggregate of 300 microseconds MD simulations recently performed on the major histocompatibility complex class II (MHCII) protein, I demonstrate how this toolkit can extract and visualize valuable mechanistic information from big MD simulation data, e.g., in form of networks of dynamic interaction changes connecting functionally relevant sites of a protein complex. AVAILABILITY AND IMPLEMENTATION: PySFD is freely available under the L-GPL license at https://github.com/markovmodel/PySFD. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Major Histocompatibility Complex (MHC) Class I and MHC Class II Proteins: Conformational Plasticity in Antigen Presentation.

Antigen presentation by major histocompatibility complex (MHC) proteins is essential for adaptive immunity. Prior to presentation, peptides need to be generated from proteins that are either produced by the cell's own translational machinery or that are funneled into the endo-lysosomal vesicular system. The prolonged interaction between a T cell receptor and specific pMHC complexes, after an extensive search process in secondary lymphatic organs, eventually triggers T cells to proliferate and to mount a specific cellular immune response. Once processed, the peptide repertoire presented by MHC proteins largely depends on structural features of the binding groove of each particular MHC allelic variant. Additionally, two peptide editors-tapasin for class I and HLA-DM for class II-contribute to the shaping of the presented peptidome by favoring the binding of high-affinity antigens. Although there is a vast amount of biochemical and structural information, the mechanism of the catalyzed peptide exchange for MHC class I and class II proteins still remains controversial, and it is not well understood why certain MHC allelic variants are more susceptible to peptide editing than others. Recent studies predict a high impact of protein intermediate states on MHC allele-specific peptide presentation, which implies a profound influence of MHC dynamics on the phenomenon of immunodominance and the development of autoimmune diseases. Here, we review the recent literature that describe MHC class I and II dynamics from a theoretical and experimental point of view and we highlight the similarities between MHC class I and class II dynamics despite the distinct functions they fulfill in adaptive immunity.

The Isomeric Preference of an Atypical Dopamine Transporter Inhibitor Contributes to Its Selection of the Transporter Conformation.

Cocaine, a widely abused psychostimulant, inhibits the dopamine transporter (DAT) by trapping the protein in an outward-open conformation, whereas atypical DAT inhibitors such as benztropine have low abuse liability and prefer less outward-open conformations. Here, we use a spectrum of computational modeling and simulation approaches to obtain the underlying molecular mechanism in atomistic detail. Interestingly, our quantum mechanical calculations and molecular dynamics (MD) simulations suggest that a benztropine derivative JHW007 prefers a different stereoisomeric conformation of tropane in binding to DAT compared to that of a cocaine derivative, CFT. To further investigate the different inhibition mechanisms of DAT, we carried out MD simulations in combination with Markov state modeling analysis of wild-type and Y156F DAT in the absence of any ligand or the presence of CFT or JHW007. Our results indicate that the Y156F mutation and CFT shift the conformational equilibrium toward an outward-open conformation, whereas JHW007 prefers an inward-occluded conformation. Our findings reveal the mechanistic details of DAT inhibition by JHW007 at the atomistic level, which provide clues for rational design of atypical inhibitors.

The role of transmembrane segment 5 (TM5) in Na2 release and the conformational transition of neurotransmitter:sodium symporters toward the inward-open state.

Neurotransmitter:sodium symporters (NSSs) terminate neurotransmission by the reuptake of released neurotransmitters. This active accumulation of substrate against its concentration gradient is driven by the transmembrane Na+ gradient and requires that the transporter traverses several conformational states. LeuT, a prokaryotic NSS homolog, has been crystallized in outward-open, outward-occluded, and inward-open states. Two crystal structures of another prokaryotic NSS homolog, the multihydrophobic amino acid transporter (MhsT) from Bacillus halodurans, have been resolved in novel inward-occluded states, with the extracellular vestibule closed and the intracellular portion of transmembrane segment 5 (TM5i) in either an unwound or a helical conformation. We have investigated the potential involvement of TM5i in binding and unbinding of Na2, i.e. the Na+ bound in the Na2 site, by carrying out comparative molecular dynamics simulations of the models derived from the two MhsT structures. We find that the helical TM5i conformation is associated with a higher propensity for Na2 release, which leads to the repositioning of the N terminus and transition to an inward-open state. By using comparative interaction network analysis, we also identify allosteric pathways connecting TM5i and the Na2 binding site to the extracellular and intracellular regions. Based on our combined computational and mutagenesis studies of MhsT and LeuT, we propose that TM5i plays a key role in Na2 binding and release associated with the conformational transition toward the inward-open state, a role that is likely to be shared across the NSS family.

MHC class II complexes sample intermediate states along the peptide exchange pathway.

The presentation of peptide-MHCII complexes (pMHCIIs) for surveillance by T cells is a well-known immunological concept in vertebrates, yet the conformational dynamics of antigen exchange remain elusive. By combining NMR-detected H/D exchange with Markov modelling analysis of an aggregate of 275 microseconds molecular dynamics simulations, we reveal that a stable pMHCII spontaneously samples intermediate conformations relevant for peptide exchange. More specifically, we observe two major peptide exchange pathways: the kinetic stability of a pMHCII's ground state defines its propensity for intrinsic peptide exchange, while the population of a rare, intermediate conformation correlates with the propensity of the HLA-DM-catalysed pathway. Helix-destabilizing mutants designed based on our model shift the exchange behaviour towards the HLA-DM-catalysed pathway and further allow us to conceptualize how allelic variation can shape an individual's MHC restricted immune response.

Computational approaches to detect allosteric pathways in transmembrane molecular machines.

Many of the functions of transmembrane proteins involved in signal processing and transduction across the cell membrane are determined by allosteric couplings that propagate the functional effects well beyond the original site of activation. Data gathered from breakthroughs in biochemistry, crystallography, and single molecule fluorescence have established a rich basis of information for the study of molecular mechanisms in the allosteric couplings of such transmembrane proteins. The mechanistic details of these couplings, many of which have therapeutic implications, however, have only become accessible in synergy with molecular modeling and simulations. Here, we review some recent computational approaches that analyze allosteric coupling networks (ACNs) in transmembrane proteins, and in particular the recently developed Protein Interaction Analyzer (PIA) designed to study ACNs in the structural ensembles sampled by molecular dynamics simulations. The power of these computational approaches in interrogating the functional mechanisms of transmembrane proteins is illustrated with selected examples of recent experimental and computational studies pursued synergistically in the investigation of secondary active transporters and GPCRs. This article is part of a Special Issue entitled: Membrane Proteins edited by J.C. Gumbart and Sergei Noskov.

Substrate-induced unlocking of the inner gate determines the catalytic efficiency of a neurotransmitter:sodium symporter.

Neurotransmitter:sodium symporters (NSSs) mediate reuptake of neurotransmitters from the synaptic cleft and are targets for several therapeutics and psychostimulants. The prokaryotic NSS homologue, LeuT, represents a principal structural model for Na(+)-coupled transport catalyzed by these proteins. Here, we used site-directed fluorescence quenching spectroscopy to identify in LeuT a substrate-induced conformational rearrangement at the inner gate conceivably leading to formation of a structural intermediate preceding transition to the inward-open conformation. The substrate-induced, Na(+)-dependent change required an intact primary substrate-binding site and involved increased water exposure of the cytoplasmic end of transmembrane segment 5. The findings were supported by simulations predicting disruption of an intracellular interaction network leading to a discrete rotation of transmembrane segment 5 and the adjacent intracellular loop 2. The magnitude of the spectroscopic response correlated inversely with the transport rate for different substrates, suggesting that stability of the intermediate represents an unrecognized rate-limiting barrier in the NSS transport mechanism.

Mechanism of the Association between Na+ Binding and Conformations at the Intracellular Gate in Neurotransmitter:Sodium Symporters.

Neurotransmitter:sodium symporters (NSSs) terminate neurotransmission by Na(+)-dependent reuptake of released neurotransmitters. Previous studies suggested that Na(+)-binding reconfigures dynamically coupled structural elements in an allosteric interaction network (AIN) responsible for function-related conformational changes, but the intramolecular pathway of this mechanism has remained uncharted. We describe a new approach for the modeling and analysis of intramolecular dynamics in the bacterial NSS homolog LeuT. From microsecond-scale molecular dynamics simulations and cognate experimental verifications in both LeuT and human dopamine transporter (hDAT), we apply the novel method to identify the composition and the dynamic properties of their conserved AIN. In LeuT, two different perturbations disrupting Na(+) binding and transport (i.e. replacing Na(+) with Li(+) or the Y268A mutation at the intracellular gate) affect the AIN in strikingly similar ways. In contrast, other mutations that affect the intracellular gate (i.e. R5A and D369A) do not significantly impair Na(+) cooperativity and transport. Our analysis shows these perturbations to have much lesser effects on the AIN, underscoring the sensitivity of this novel method to the mechanistic nature of the perturbation. Notably, this set of observations holds as well for hDAT, where the aligned Y335A, R60A, and D436A mutations also produce different impacts on Na(+) dependence. Thus, the detailed AIN generated from our method is shown to connect Na(+) binding with global conformational changes that are critical for the transport mechanism. That the AIN between the Na(+) binding sites and the intracellular gate in bacterial LeuT resembles that in eukaryotic hDAT highlights the conservation of allosteric pathways underlying NSS function.

Transport domain unlocking sets the uptake rate of an aspartate transporter.

Glutamate transporters terminate neurotransmission by clearing synaptically released glutamate from the extracellular space, allowing repeated rounds of signalling and preventing glutamate-mediated excitotoxicity. Crystallographic studies of a glutamate transporter homologue from the archaeon Pyrococcus horikoshii, GltPh, showed that distinct transport domains translocate substrates into the cytoplasm by moving across the membrane within a central trimerization scaffold. Here we report direct observations of these 'elevator-like' transport domain motions in the context of reconstituted proteoliposomes and physiological ion gradients using single-molecule fluorescence resonance energy transfer (smFRET) imaging. We show that GltPh bearing two mutations introduced to impart characteristics of the human transporter exhibits markedly increased transport domain dynamics, which parallels an increased rate of substrate transport, thereby establishing a direct temporal relationship between transport domain motion and substrate uptake. Crystallographic and computational investigations corroborated these findings by revealing that the 'humanizing' mutations favour structurally 'unlocked' intermediate states in the transport cycle exhibiting increased solvent occupancy at the interface between the transport domain and the trimeric scaffold.

Chloride binding site of neurotransmitter sodium symporters.

Neurotransmitter:sodium symporters (NSSs) play a critical role in signaling by reuptake of neurotransmitters. Eukaryotic NSSs are chloride-dependent, whereas prokaryotic NSS homologs like LeuT are chloride-independent but contain an acidic residue (Glu290 in LeuT) at a site where eukaryotic NSSs have a serine. The LeuT-E290S mutant displays chloride-dependent activity. We show that, in LeuT-E290S cocrystallized with bromide or chloride, the anion is coordinated by side chain hydroxyls from Tyr47, Ser290, and Thr254 and the side chain amide of Gln250. The bound anion and the nearby sodium ion in the Na1 site organize a connection between their coordinating residues and the extracellular gate of LeuT through a continuous H-bond network. The specific insights from the structures, combined with results from substrate binding studies and molecular dynamics simulations, reveal an anion-dependent occlusion mechanism for NSS and shed light on the functional role of chloride binding.

Ion-controlled conformational dynamics in the outward-open transition from an occluded state of LeuT.

Neurotransmitter:sodium symporter (NSS) proteins are secondary Na(+)-driven active transporters that terminate neurotransmission by substrate uptake. Despite the availability of high-resolution crystal structures of a bacterial homolog of NSSs-Leucine Transporter (LeuT)-and extensive computational and experimental structure-function studies, unanswered questions remain regarding the transport mechanisms. We used microsecond atomistic molecular-dynamics (MD) simulations and free-energy computations to reveal ion-controlled conformational dynamics of LeuT in relation to binding affinity and selectivity of the more extracellularly positioned Na(+) binding site (Na1 site). In the course of MD simulations starting from the occluded state with bound Na(+), but in the absence of substrate, we find a spontaneous transition of the extracellular vestibule of LeuT into an outward-open conformation. The outward opening is enhanced by the absence of Na1 and modulated by the protonation state of the Na1-associated Glu-290. Consistently, the Na(+) affinity for the Na1 site is inversely correlated with the extent of outward-open character and is lower than in the occluded state with bound substrate; however, the Na1 site retains its selectivity for Na(+) over K(+) in such conformational transitions. To the best of our knowledge, our findings shed new light on the Na(+)-driven transport cycle and on the symmetry in structural rearrangements for outward- and inward-open transitions.

Structural intermediates in a model of the substrate translocation path of the bacterial glutamate transporter homologue GltPh.

Excitatory amino acid transporters (EAATs) are membrane proteins responsible for reuptake of glutamate from the synaptic cleft to terminate neurotransmission and help prevent neurotoxically high, extracellular glutamate concentrations. Important structural information about these proteins emerged from crystal structures of GltPh, a bacterial homologue of EAATs, in conformations facing outward and inward. These remarkably different conformations are considered to be end points of the substrate translocation path (STP), suggesting that the transport mechanism involves major conformational rearrangements that remain uncharted. To investigate possible steps in the structural transitions of the STP between the two end-point conformations, we applied a combination of computational modeling methods (motion planning, molecular dynamics simulations, and mixed elastic network models). We found that the conformational changes in the transition involve mainly the repositioning the "transport domain" and the "trimerization domain" identified previously in the crystal structures. The two domains move in opposite directions along the membrane normal, and the transport domain also tilts by ∼17° with respect to this axis. Moreover, the TM3-4 loop undergoes a flexible, "restraining bar"-like conformational change with respect to the transport domain. As a consequence of these conformational rearrangements along the transition path we calculated a significant decrease of nearly 20% in the area of the transport-to-trimerization domain interface (TTDI). Water penetrates parts of the TTDI in the modeled intermediates but very much less in the end-point conformations. We show that these characteristics of the modeled intermediate states agree with experimental results from residue-accessibility studies in individual monomers and identify specific residues that can be used to test the proposed STP. Moreover, MD simulations of complete GltPh trimers constructed from initially identical monomer intermediates suggest that asymmetry can appear in the trimer, consonant with available experimental data showing independent transport kinetics by individual monomers in the trimers.

A dynamic model of membrane-bound phospholipase Cß2 activation by Gßγ subunits.

Phospholipase C (PLC) ß2, a well studied member of the family of enzymes that catalyze the hydrolysis of the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) into secondary messengers, can be activated by the Gßγ subunits of heterotrimeric G-proteins in a manner that depends on the presence and composition of the associated phospholipid membrane surface. The N-terminal pleckstrin homology (PH) domain of PLCß2 mediates both the response to Gßγ and membrane binding, but how these interactions are coupled to yield an activated catalytic core remains unknown. Here we propose a mechanism based on molecular models of truncated PLCß2 in its activated form complexed with Gßγ and in the catalytically inactive/membrane-bound form, obtained with the application of protein-protein docking algorithms and coarse-grained molecular dynamics simulations. These models were probed experimentally, and the inferences were confirmed by results from a combination of molecular biology and fluorescence assays. Results from the dynamic simulations of the molecular models and their interactions with various lipid bilayers identify the determinants of PLCß2-PH domain specificity for Gßγ and lipid membranes and suggest a mechanism for the previously reported dependence of Gßγ activation on the associated membrane composition. Together, these findings explain the roles of the different activators in terms of their effect on the orientations of the PH and catalytic core domains relative to the lipid membranes.