Interactions in native binding sites cause a large change in protein dynamics

Cellular functions are regulated by molecules that interact with proteins and alter their activities. To enable such control, protein activity, and therefore protein conformational distributions, must be susceptible to alteration by molecular interactions at functional sites. Here we investigate whether interactions at functional sites cause a large change in the protein conformational distribution. We apply a computational method, called dynamics perturbation analysis (DPA), to identify sites at which interactions have a large allosteric potential D(x), which is the Kullback-Leibler divergence between protein conformational distributions with and without an interaction. In DPA, a protein is decorated with surface points that interact with neighboring protein atoms, and D(x) is calculated for each of the points in a coarse-grained model of protein vibrations. We use DPA to examine hundreds of protein structures from a standard small-molecule docking test set, and find that ligand-binding sites have elevated values of D(x): for 95% of proteins, the probability of randomly obtaining values as high as those in the binding site is 10(-3) or smaller. We then use DPA to develop a computational method to predict functional sites in proteins, and find that the method accurately predicts ligand-binding-site residues for proteins in the test set. The performance of this method compares favorably with that of a cleft analysis method. The results confirm that interactions at small-molecule binding sites cause a large change in the protein conformational distribution, and motivate using DPA for large-scale prediction of functional sites in proteins. They also suggest that natural selection favors proteins whose activities are capable of being regulated by molecular interactions.     

An automated immunoassay for early specificity profiling of antibodies

Antibody-based therapeutics are of great value for the treatment of human diseases. In addition to functional activity, affinity or physico-chemical properties, antibody specificity is considered to be one of the most crucial attributes for safety and efficacy. Consequently, appropriate studies are required before entering clinical trials.

High content protein arrays are widely applied to assess antibody specificity, but this commercial solution can only be applied to final therapeutic antibody candidates because such arrays are expensive and their throughput is limited. A flexible, high-throughput and economical assay that allows specificity testing of IgG or Fab molecules during early discovery is described here. The 384-well microtiter plate assay contains a comprehensive panel of 32 test proteins and uses electrochemiluminescence as readout.

The Protein Panel Profiling (3P) was used to analyze marketed therapeutic antibodies that all showed highly specific binding profiles. Subsequently, 3P was applied to antibody candidates from early discovery and the results compared well with those obtained with a commercially available high content protein chip. Our results suggest that 3P can be applied as an additional filter for lead selection, allowing the identification of favorable antibody candidates in early discovery and thereby increasing the speed and possibility of success in drug development.     

Conformational states of the Rapana thomasiana hemocyanin and its substructures studied by dynamic light scattering and time-resolved fluorescence spectroscopy

Hemocyanins are dioxygen-transporting proteins freely dissolved in the hemolymph of mollusks and arthropods. Dynamic light scattering and time-resolved fluorescence measurements show that the oxygenated and apo-forms of the Rapana thomasiana hemocyanin, its structural subunits RtH1 and RtH2, and those of the functional unit RtH2e, exist in different conformations. The oxygenated respiratory proteins are less compact and more asymmetric than the respective apo-forms. Different conformational states were also observed for the R. thomasiana hemocyanin in the absence and presence of an allosteric regulator. The results are in agreement with a molecular mechanism for cooperative dioxygen binding in molluscan hemocyanins including transfer of conformational changes from one functional unit to another.     

Association of Proteins with the Stress 70 Molecular Chaperones at Low Temperature: Evidence for the Existence of Cold Labile Proteins in Spinach

Molecular chaperones of the stress 70 family reversibly bind and release nonnative proteins in a nucleotide-dependent cycle. Purified monoclonal antibodies prepared against spinach (Spinacia oleracea) stress 70 molecular chaperones were used in immunoprecipitation experiments with extracts of spinach leaf tissue pulse-labeled with [³⁵S]methionine in an effort to detect whether low-temperature exposure altered the biogenesis or the native state stability of any proteins leading to the formation of complexes with the stress 70 molecular chaperones. The two monoclonal antibodies used in this research are highly specific for the cytosolic or ER-luminal stress 70 molecular chaperones. Analyses of the immunoprecipitation results indicate that low temperature causes an increased association of some proteins with the two chaperones. The findings are consistent with the hypothesis that normal biogenesis or the conformational stability of specific proteins may be unfavorably altered at low temperature in spinach and perhaps other plants.     

Hierarchical Conformational Analysis of Native Lysozyme Based on Sub-Millisecond Molecular Dynamics Simulations

Hierarchical organization of free energy landscape (FEL) for native globular proteins has been widely accepted by the biophysics community. However, FEL of native proteins is usually projected onto one or a few dimensions. Here we generated collectively 0.2 milli-second molecular dynamics simulation trajectories in explicit solvent for hen egg white lysozyme (HEWL), and carried out detailed conformational analysis based on backbone torsional degrees of freedom (DOF). Our results demonstrated that at micro-second and coarser temporal resolutions, FEL of HEWL exhibits hub-like topology with crystal structures occupying the dominant structural ensemble that serves as the hub of conformational transitions. However, at 100ns and finer temporal resolutions, conformational substates of HEWL exhibit network-like topology, crystal structures are associated with kinetic traps that are important but not dominant ensembles. Backbone torsional state transitions on time scales ranging from nanoseconds to beyond microseconds were found to be associated with various types of molecular interactions. Even at nanoseconds temporal resolution, the number of conformational substates that are of statistical significance is quite limited. These observations suggest that detailed analysis of conformational substates at multiple temporal resolutions is both important and feasible. Transition state ensembles among various conformational substates at microsecond temporal resolution were observed to be considerably disordered. Life times of these transition state ensembles are found to be nearly independent of the time scales of the participating torsional DOFs.     

Myosin and Tropomyosin Stabilize the Conformation of Formin-nucleated Actin Filaments

The conformational elasticity of the actin cytoskeleton is essential for its versatile biological functions. Increasing evidence supports that the interplay between the structural and functional properties of actin filaments is finely regulated by actin-binding proteins; however, the underlying mechanisms and biological consequences are not completely understood. Previous studies showed that the binding of formins to the barbed end induces conformational transitions in actin filaments by making them more flexible through long range allosteric interactions. These conformational changes are accompanied by altered functional properties of the filaments. To get insight into the conformational regulation of formin-nucleated actin structures, in the present work we investigated in detail how binding partners of formin-generated actin structures, myosin and tropomyosin, affect the conformation of the formin-nucleated actin filaments using fluorescence spectroscopic approaches. Time-dependent fluorescence anisotropy and temperature-dependent Förster-type resonance energy transfer measurements revealed that heavy meromyosin, similarly to tropomyosin, restores the formin-induced effects and stabilizes the conformation of actin filaments. The stabilizing effect of heavy meromyosin is cooperative. The kinetic analysis revealed that despite the qualitatively similar effects of heavy meromyosin and tropomyosin on the conformational dynamics of actin filaments the mechanisms of the conformational transition are different for the two proteins. Heavy meromyosin stabilizes the formin-nucleated actin filaments in an apparently single step reaction upon binding, whereas the stabilization by tropomyosin occurs after complex formation. These observations support the idea that actin-binding proteins are key elements of the molecular mechanisms that regulate the conformational and functional diversity of actin filaments in living cells.     

Neutralizing Antibodies Against Allosteric Proteins: Insights From a Bacterial Adhesin

Allosteric proteins transition between ‘inactive’ and ‘active’ states. In general, such proteins assume distinct conformational states at the level of secondary, tertiary and/or quaternary structure. Different conformers of an allosteric protein can be antigenically dissimilar and induce antibodies with a highly distinctive specificities and neutralizing functional effects. Here we summarize studies on various functional types of monoclonal antibodies obtained against different allosteric conformers of the mannose-specific bacterial adhesin FimH – the most common cell attachment protein of Escherichia coli and other enterobacterial pathogens. Included are types of antibodies that activate the FimH function via interaction with ligand-induced binding sites or by wedging between domains as well as antibodies that inhibit FimH through orthosteric, parasteric, or novel dynasteric mechanisms. Understanding the molecular mechanism of antibody action against allosteric proteins provides insights on how to design antibodies with a desired functional effect, including those with neutralizing activity against bacterial and viral cell attachment proteins.     

Cooperative binding and activation of fibronectin by a bacterial surface protein

Integrin-dependent cell invasion of some pathogenic bacteria is mediated by surface proteins targeting the extracellular matrix protein fibronectin (FN). Although the structural basis for bacterial FN recognition is well understood, it has been unclear why proteins such as streptococcal SfbI contain several FN-binding sites. We used microcalorimetry to reveal cooperative binding of FN fragments to arrays of binding sites in SfbI. In combination with thermodynamic analyses, functional cell-based assays show that SfbI induces conformational changes in the N-terminal 100-kDa region of FN (FN100kDa), most likely by competition with intramolecular interactions defining an inactive state of FN100kDa. This study provides insights into how long range conformational changes resulting in FN activation may be triggered by bacterial pathogens.     

Arrestins: structural disorder creates rich functionality

Arrestins are soluble relatively small 44–46 kDa proteins that specifically bind hundreds of active phosphorylated GPCRs and dozens of non-receptor partners. There are binding partners that demonstrate preference for each of the known arrestin conformations: free, receptor-bound, and microtubule-bound. Recent evidence suggests that conformational flexibility in every functional state is the defining characteristic of arrestins. Flexibility, or plasticity, of proteins is often described as structural disorder, in contrast to the fixed conformational order observed in high-resolution crystal structures. However, protein-protein interactions often involve highly flexible elements that can assume many distinct conformations upon binding to different partners. Existing evidence suggests that arrestins are no exception to this rule: their flexibility is necessary for functional versatility. The data on arrestins and many other multi-functional proteins indicate that in many cases, “order” might be artificially imposed by highly non-physiological crystallization conditions and/or crystal packing forces. In contrast, conformational flexibility (and its extreme case, intrinsic disorder) is a more natural state of proteins, representing true biological order that underlies their physiologically relevant functions.     

The dynamics of camphor in the cytochrome P450 CYP101D2

The recent crystal structures of CYP101D2, a cytochrome P450 protein from the oligotrophic bacterium Novosphingobium aromaticivorans DSM12444 revealed that both the native (substrate‐free) and camphor‐soaked forms have open conformations. Furthermore, two other potential camphor‐binding sites were also identified from electron densities in the camphor‐soaked structure, one being located in the access channel and the other in a cavity on the surface near the F‐helix side of the F‐G loop termed the substrate recognition site. These latter sites may be key intermediate positions on the pathway for substrate access to or product egress from the active site. Here, we show via the use of unbiased atomistic molecular dynamics simulations that despite the open conformation of the native and camphor‐bound crystal structures, the underlying dynamics of CYP101D2 appear to be very similar to other CYP proteins. Simulations of the native structure demonstrated that the protein is capable of sampling many different conformational substates. At the same time, simulations with the camphor positioned at various locations within the access channel or recognition site show that movement towards the active site or towards bulk solvent can readily occur on a short timescale, thus confirming many previously reported in silico studies using steered molecular dynamics. The simulations also demonstrate how the fluctuations of an aromatic gate appear to control access to the active site. Finally, comparison of camphor‐bound simulations with the native simulations suggests that the fluctuations can be of similar level and thus are more representative of the conformational selection model rather than induced fit.     

The myotubularin–amphiphysin 2 complex in membrane tubulation and centronuclear myopathies

Myotubularin (MTM1) and amphiphysin 2 (BIN1) are two proteins mutated in different forms of centronuclear myopathy, but the functional and pathological relationship between these two proteins was unknown. Here, we identified MTM1 as a novel binding partner of BIN1, both in vitro and endogenously in skeletal muscle. Moreover, MTM1 enhances BIN1‐mediated membrane tubulation, depending on binding and phosphoinositide phosphatase activity. BIN1 patient mutations induce a conformational change in BIN1 and alter its binding and regulation by MTM1. In conclusion, we identified the first molecular and functional link between MTM1 and BIN1, supporting a common pathological mechanism in different forms of centronuclear myopathy.     

Criticality in the conformational phase transition among self-similar groups in intrinsically disordered proteins: Probed by salt-bridge dynamics

Intrinsically disordered proteins (IDP) serve as one of the key components in the global proteome. In contrast to globular proteins, they harbor an enormous amount of physical flexibility enforcing them to be retained in conformational ensembles rather than stable folds. Previous studies in an aligned direction have revealed the importance of transient dynamical phenomena like that of salt-bridge formation in IDPs to support their physical flexibility and have further highlighted their functional relevance. For this characteristic flexibility, IDPs remain amenable and accessible to different ordered binding partners, supporting their potential multi-functionality. The current study further addresses this complex structure-functional interplay in IDPs using phase transition dynamics to conceptualize the underlying (avalanche type) mechanism of their being distributed across and hopping around degenerate structural states (conformational ensembles). For this purpose, extensive molecular dynamics simulations have been done and the data analyzed from a statistical physics perspective. Investigation of the plausible scope of 'self-organized criticality' (SOC) to fit into the complex dynamics of IDPs was found to be assertive, relating the conformational degeneracy of these proteins to their functional multiplicity. In accordance with the transient nature of 'salt-bridge dynamics', the study further uses it as a probe to explain the structural basis of the proposed criticality in the conformational phase transition among self-similar groups in IDPs. The analysis reveal scale-invariant self-similar fractal geometries in the structural conformations of different IDPs. The insights from the study has the potential to be extended further to benefit structural tinkering of IDPs in their functional characterization and drugging.     

Tracking Molecular Interactions in Membranes by Simultaneous ATR-FTIR-AFM

In situ atomic force microscopy (AFM) is an exceedingly powerful and useful technique for characterizing the structure and assembly of proteins in real-time, in situ, and especially at model membrane interfaces, such as supported planar lipid bilayers. There remains, however, a fundamental challenge with AFM-based imaging. Conclusions are inferred based on morphological or topographical features. It is conventionally very difficult to use AFM to confirm specific molecular conformation, especially in the case of protein-membrane interactions. In this case, a protein may undergo subtle conformational changes upon insertion in the membrane that may be critical to its function. AFM lacks the ability to directly measure such conformational changes and can, arguably, only resolve features that are topographically distinct. To address these issues, we have developed a platform that integrates in situ AFM with attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy. This combination of tools provides a unique means of tracking, simultaneously, conformational changes, not resolvable by in situ AFM, with topographical details that are not readily identified by conventional spectroscopy. Preliminary studies of thermal transitions in supported lipid bilayers and direct evidence of lipid-induced conformational changes in adsorbed proteins illustrates the potential of this coupled in situ functional imaging strategy.     

Analysis of Chaperone Function and Formation of Hetero-oligomeric Complexes of Hsp18.1 and Hsp17.7, Representing Two Different Cytoplasmic sHSP Classes in Pisum sativum

Small heat shock proteins (sHSPs) are the most abundant stress proteins in plants. Usually not expressed under permissive conditions, they can accumulate to more than 2% of the total cellular protein content during heat stress. At present several points of evidence indicate that these proteins act as molecular chaperones by keeping partially denatured proteins in a folding-competent state. In plants sHSPs are encoded by a multigene family, which can be segregated into several classes according to their subcellular position and/or sequence homology. Curiously, two different classes appear in the cytoplasm. Their specific role during heat shock remains elusive. Here we present some evidence that both classes of sHSPs enhance recovery of reporter protein activity in the presence of HSP70. Applying peptide arrays prepared by SPOT synthesis and in situ analysis by confocal laser scanning microscopy, we could further show that the two classes of sHSP are attached to each other and are able to interact with non-native proteins both in vivo and in vitro. Although both of the sHSPs act similarly as molecular chaperones, immunohistochemistry experiments support the hypothesis that the two have different cellular functions in the development of heat-induced cytoplasmic heat shock granules under elevated temperatures. Daniela Wagner Deceased 24 Feburary 2004.     

Loss of intramolecular electrostatic interactions and limited conformational ensemble may promote self‐association of cis–tau peptide

Self‐association of proteins can be triggered by a change in the distribution of the conformational ensemble. Posttranslational modification, such as phosphorylation, can induce a shift in the ensemble of conformations. In the brain of Alzheimer's disease patients, the formation of intra‐cellular neurofibrillary tangles deposition is a result of self‐aggregation of hyper‐phosphorylated tau protein. Biochemical and NMR studies suggest that the cis peptidyl prolyl conformation of a phosphorylated threonine‐proline motif in the tau protein renders tau more prone to aggregation than the trans isomer. However, little is known about the role of peptidyl prolyl cis/trans isomerization in tau aggregation. Here, we show that intra‐molecular electrostatic interactions are better formed in the trans isomer. We explore the conformational landscape of the tau segment containing the phosphorylated‐Thr²³¹‐Pro²³² motif using accelerated molecular dynamics and show that intra‐molecular electrostatic interactions are coupled to the isomeric state of the peptidyl prolyl bond. Our results suggest that the loss of intra‐molecular interactions and the more restricted conformational ensemble of the cis isomer could favor self‐aggregation. The results are consistent with experiments, providing valuable complementary atomistic insights and a hypothetical model for isomer specific aggregation of the tau protein. Proteins 2015; 83:436–444. © 2014 Wiley Periodicals, Inc.     

Conformational energy analysis of the leucine repeat regions of C/EBP,GCN4, and the proteins of themyc,jun, andfos oncogenes

It has been recently proposed that certain DNA binding proteins (including C/EBP, GCN4 and themyc, jun, andfos oncogene proteins) share a common structural motif based on helix-promoting regions containing heptad repeat sequences of leucines. It has been suggested that this structure is critical to the biological activity of these proteins, since it facilitates the formation of functional dimers held together by interdigitating leucine side-chains along the hydrophobic interfaces between long α-helical regions of the polypeptide chains in a configuration termed the “leucine zipper.” In this paper, conformational energy analysis is used to determine the preferred three-dimensional structures of the leucine repeat regions of these proteins. The results indicate that, in all cases, the global minimum energy conformation for these regions is an amphipathic α-helix with the leucine side-chains arrayed on one side in such a way to favor “leucine zipper” dimerization. Furthermore, amino acid substitutions in these regions (such as Pro for Leu), that are known to inhibit dimer formation and prevent DNA binding, are found to produce significant conformational changes that disrupt the amphipathic helical structure. Thus, these results provide support for the proposed “leucine zipper” configuration as a critical structural feature of this class of DNA binding proteins.

     

Unraveling the dynamics of protein interactions with quantitative mass spectrometry

Knowledge of structure and dynamics of proteins and protein complexes is important to unveil the molecular basis and mechanisms involved in most biological processes. Protein complex dynamics can be defined as the changes in the composition of a protein complex during a cellular process. Protein dynamics can be defined as conformational changes in a protein during enzyme activation, for example, when a protein binds to a ligand or when a protein binds to another protein. Mass spectrometry (MS) combined with affinity purification has become the analytical tool of choice for mapping protein–protein interaction networks and the recent developments in the quantitative proteomics field has made it possible to identify dynamically interacting proteins. Furthermore, hydrogen/deuterium exchange MS is emerging as a powerful technique to study structure and conformational dynamics of proteins or protein assemblies in solution. Methods have been developed and applied for the identification of transient and/or weak dynamic interaction partners and for the analysis of conformational dynamics of proteins or protein complexes. This review is an overview of existing and recent developments in studying the overall dynamics of in vivo protein interaction networks and protein complexes using MS-based methods.     

Amyloid properties of the yeast cell wall protein Toh1 and its interaction with prion proteins Rnq1 and Sup35

Amyloids are non-branching fibrils that are composed of stacked monomers stabilized by intermolecular β-sheets. Some amyloids are associated with incurable diseases, whereas others, functional amyloids, regulate different vital processes. The prevalence and significance of functional amyloids in wildlife are still poorly understood. In recent years, by applying new approach of large-scale proteome screening, a number of novel candidate amyloids were identified in the yeast Saccharomyces cerevisiae, many of which are localized in the yeast cell wall. In this work, we showed that one of these proteins, Toh1, possess amyloid properties. The Toh1-YFP hybrid protein forms detergent-resistant aggregates in the yeast cells while being expressed under its own P_TOH1 or inducible P_CUP1 promoter. Using bacterial system for generation of extracellular amyloid aggregates C-DAG, we demonstrated that the N-terminal Toh1 fragment, containing amyloidogenic regions predicted in silico, binds Congo Red dye, manifests ‘apple-green’ birefringence when examined between crossed polarizers, and forms amyloid-like fibrillar aggregates visualized by TEM. We have established that the Toh1(20–365)-YFP hybrid protein fluorescent aggregates are co-localized with a high frequency with Rnq1C-CFP and Sup35NM-CFP aggregates in the yeast cells containing [PIN⁺] and [PSI⁺] prions, and physical interaction of these aggregated proteins was confirmed by FRET. This is one of a few known cases of physical interaction of non-Q/N-rich amyloid-like protein and Q/N-rich amyloids, suggesting that interaction of different amyloid proteins may be determined not only by similarity of their primary structures but also by similarity of their secondary structures and of conformational folds.     

Conformational selection in protein binding and function

Protein binding and function often involves conformational changes. Advanced nuclear magnetic resonance (NMR) experiments indicate that these conformational changes can occur in the absence of ligand molecules (or with bound ligands), and that the ligands may “select” protein conformations for binding (or unbinding). In this review, we argue that this conformational selection requires transition times for ligand binding and unbinding that are small compared to the dwell times of proteins in different conformations, which is plausible for small ligand molecules. Such a separation of timescales leads to a decoupling and temporal ordering of binding/unbinding events and conformational changes. We propose that conformational‐selection and induced‐change processes (such as induced fit) are two sides of the same coin, because the temporal ordering is reversed in binding and unbinding direction. Conformational‐selection processes can be characterized by a conformational excitation that occurs prior to a binding or unbinding event, while induced‐change processes exhibit a characteristic conformational relaxation that occurs after a binding or unbinding event. We discuss how the ordering of events can be determined from relaxation rates and effective on‐ and off‐rates determined in mixing experiments, and from the conformational exchange rates measured in advanced NMR or single‐molecule fluorescence resonance energy transfer experiments. For larger ligand molecules such as peptides, conformational changes and binding events can be intricately coupled and exhibit aspects of conformational‐selection and induced‐change processes in both binding and unbinding direction.