Publication date: 9 October 2015
Source:Journal of Molecular Biology, Volume 427, Issue 20

Author(s): Misbha Ud Din Ahmad, Ingrid Waege, Winfried Hausner, Michael Thomm, Winfried Boos, Kay Diederichs, Wolfram Welte

The crystal structure of TrmBL2 from the archaeon Pyrococcus furiosus shows an association of two pseudosymmetric dimers. The dimers follow the prototypical design of known bacterial repressors with two helix–turn–helix (HTH) domains binding to successive major grooves of the DNA. However, in TrmBL2, the two dimers are arranged at a mutual displacement of approximately 2bp so that they associate with the DNA along the double-helical axis at an angle of approximately 80°. While the deoxyribose phosphate groups of the double-stranded DNA (dsDNA) used for co-crystallization are clearly seen in the electron density map, most of the nucleobases are averaged out. Refinement required to assume a superposition of at least three mutually displaced dsDNAs. The HTH domains interact primarily with the deoxyribose phosphate groups and polar interactions with the nucleobases are almost absent. This hitherto unseen mode of DNA binding by TrmBL2 seems to arise from nonoptimal protein–DNA contacts made by its four HTH domains resulting in a low-affinity, nonspecific binding to DNA.
Graphical abstract

Publication date: 9 October 2015
Source:Journal of Molecular Biology, Volume 427, Issue 20

Author(s): Sojin An, Hanseong Kim, Uhn-Soo Cho

CENP-A is a centromere-specific histone H3 variant that is required for kinetochore assembly and accurate chromosome segregation. For it to function properly, CENP-A must be specifically localized to centromeres. In fission yeast, Scm3sp and the Mis18 complex, composed of Mis16, Eic1, and Mis18, function as a CENP-ACnp1-specific chaperone and a recruiting factor, respectively, and together ensure accurate delivery of CENP-ACnp1 to centromeres. Although how Scm3sp specifically recognizes CENP-ACnp1 has been revealed recently, the recruiting mechanism of CENP-ACnp1 via the Mis18 complex remains unknown. In this study, we have determined crystal structures of Schizosaccharomyces japonicus Mis16 alone and in complex with the helix 1 of histone H4 (H4α1). Crystal structures followed by mutant analysis and affinity pull-downs have revealed that Mis16 recognizes both H4α1 and Scm3sp independently within the CENP-ACnp1/H4:Scm3sp complex. This observation suggests that Mis16 gains CENP-ACnp1 specificity by recognizing both Scm3sp and histone H4. Our studies provide insights into the molecular mechanisms underlying specific recruitment of CENP-ACnp1/H4:Scm3sp into centromeres.
Graphical abstract

Publication date: 9 October 2015
Source:Journal of Molecular Biology, Volume 427, Issue 20

Author(s): Christopher E. Morgan, Jennifer L. Meagher, Jeffrey D. Levengood, James Delproposto, Carrie Rollins, Jeanne A. Stuckey, Blanton S. Tolbert

The heterogeneous nuclear ribonucleoprotein (hnRNP) A1 protein is a multifunctional RNA binding protein implicated in a wide range of biological functions. Mechanisms and putative hnRNP A1–RNA interactions have been inferred primarily from the crystal structure of its UP1 domain bound to ssDNA. RNA stem loops represent an important class of known hnRNP A1 targets, yet little is known about the structural basis of hnRNP A1–RNA recognition. Here, we report the first high-resolution structure (1.92Å) of UP1 bound to a 5′-AGU-3′ trinucleotide that resembles sequence elements of several native hnRNP A1–RNA stem loop targets. UP1 interacts specifically with the AG dinucleotide sequence via a “nucleobase pocket” formed by the β-sheet surface of RRM1 and the inter-RRM linker; RRM2 does not contact the RNA. The inter-RRM linker forms the lid of the nucleobase pocket and we show using structure-guided mutagenesis that the conserved salt-bridge interactions (R75:D155 and R88:D157) on the α-helical side of the RNA binding surface stabilize the linker in a geometry poised to bind RNA. We further investigated the structural basis of UP1 binding HIViSL3ESS3 by determining a structural model of the complex scored by small-angle X-ray scattering. UP1 docks on the apical loop of SL3ESS3 using its RRM1 domain and inter-RRM linker only. The biophysical implications of the structural model were tested by measuring kinetic binding parameters, where mutations introduced within the apical loop reduce binding affinities by slowing down the rate of complex formation. Collectively, the data presented here provide the first insights into hnRNP A1–RNA interactions.
Graphical abstract

Publication date: 9 October 2015
Source:Journal of Molecular Biology, Volume 427, Issue 20

Author(s): Ignacio Fernández, Lisandro H. Otero, Sebastián Klinke, Mariela del Carmen Carrica, Fernando A. Goldbaum

Brucella abortus is an important pathogenic bacterium that has to overcome oxygen deficiency in order to achieve a successful infection. Previously, we proved that a two-component system formed by the histidine kinase NtrY and the response regulator NtrX is essential to achieve an adaptive response to low oxygen tension conditions. Even though the relevance of this signaling pathway has already been demonstrated in other microorganisms, its molecular activation mechanism has not yet been described in detail. In this article, we report the first crystal structures from different conformations of the NtrX receiver domain from B. abortus, and we propose a sequence of events to explain the structural rearrangements along the activation process. The analysis of the structures obtained in the presence of the phosphoryl group analog beryllofluoride led us to postulate that changes in the interface formed by the α4 helix and the β5 strand are important for the activation, producing a reorientation of the α5 helix. Also, a biochemical characterization of the NtrX receiver domain enzymatic activities was performed, describing its autophosphorylation and autodephosphorylation kinetics. Finally, the role of H85, an important residue, was addressed by site-directed mutagenesis. Overall, these results provide significant structural basis for understanding the response regulator activation in this bacterial two-component system.
Graphical abstract

Publication date: 9 October 2015
Source:Journal of Molecular Biology, Volume 427, Issue 20

Author(s): Clayton Smith, Lee Dolat, Dimitrios Angelis, Eva Forgacs, Elias T. Spiliotis, Vitold E. Galkin

Septins are a highly conserved family of proteins in eukaryotes that is recognized as a novel component of the cytoskeleton. Septin 9 (SEPT9) interacts directly with actin filaments and functions as an actin stress fiber cross-linking protein that promotes the maturation of nascent focal adhesions and cell migration. However, the molecular details of how SEPT9 interacts with F-actin remain unknown. Here, we use electron microscopy and image analysis to show that SEPT9 binds to F-actin in a highly polymorphic fashion. We demonstrate that the basic domain (B-domain) of the N-terminal tail of SEPT9 is responsible for actin cross-linking, while the GTP-binding domain (G-domain) does not bundle F-actin. We show that the B-domain of SEPT9 binds to three sites on F-actin, and the two of these sites overlap with the binding regions of myosin and cofilin. SEPT9 inhibits actin-dependent ATPase activity of myosin and competes with the weakly bound state of myosin for binding to F-actin. At the same time, SEPT9 significantly reduces the extent of F-actin depolymerization by cofilin. Taken together, these data suggest that SEPT9 protects actin filaments from depolymerization by cofilin and myosin and indicate a mechanism by which SEPT9 could maintain the integrity of growing and contracting actin filaments.
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Abstract

Ubiquitin (Ub) chains regulate a wide range of biological processes, and Ub chain connectivity is a critical determinant of the many regulatory roles that this post-translational modification plays in cells. To understand how distinct Ub chains orchestrate different biochemical events, we and other investigators have developed enzymatic and non-enzymatic methods to synthesize Ub chains of well-defined length and connectivity. A number of chemical approaches have been used to generate Ub oligomers connected by non-native linkages; however, few studies have examined the extent to which non-native linkages recapitulate the structural and functional properties associated with native isopeptide bonds. Here, we compare the structure and function of Ub dimers bearing native and non-native linkages. Using small-angle X-ray scattering (SAXS) analysis, we show that scattering profiles for the two types of dimers are similar. Moreover, using an experimental structural library and atomistic simulations to fit the experimental SAXS profiles, we find that the two types of Ub dimers can be matched to analogous structures. An important application of non-native Ub oligomers is to probe the activity and selectivity of deubiquitinases. Through steady-state kinetic analyses, we demonstrate that different families of deubiquitinases hydrolyze native and non-native isopeptide linkages with comparable efficiency and selectivity. Considering the significant challenges associated with building topologically diverse native Ub chains, our results illustrate that chains harboring non-native linkages can serve as surrogate substrates for explorations of Ub function. This article is protected by copyright. All rights reserved.

by Amarda Shehu, Ruth Nussinov

by Jeffrey R. Brender, Yang Zhang

The formation of protein-protein complexes is essential for proteins to perform their physiological functions in the cell. Mutations that prevent the proper formation of the correct complexes can have serious consequences for the associated cellular processes. Since experimental determination of protein-protein binding affinity remains difficult when performed on a large scale, computational methods for predicting the consequences of mutations on binding affinity are highly desirable. We show that a scoring function based on interface structure profiles collected from analogous protein-protein interactions in the PDB is a powerful predictor of protein binding affinity changes upon mutation. As a standalone feature, the differences between the interface profile score of the mutant and wild-type proteins has an accuracy equivalent to the best all-atom potentials, despite being two orders of magnitude faster once the profile has been constructed. Due to its unique sensitivity in collecting the evolutionary profiles of analogous binding interactions and the high speed of calculation, the interface profile score has additional advantages as a complementary feature to combine with physics-based potentials for improving the accuracy of composite scoring approaches. By incorporating the sequence-derived and residue-level coarse-grained potentials with the interface structure profile score, a composite model was constructed through the random forest training, which generates a Pearson correlation coefficient >0.8 between the predicted and observed binding free-energy changes upon mutation. This accuracy is comparable to, or outperforms in most cases, the current best methods, but does not require high-resolution full-atomic models of the mutant structures. The binding interface profiling approach should find useful application in human-disease mutation recognition and protein interface design studies.

by Xiongwu Wu, Bernard R. Brooks

Chemical and thermodynamic equilibrium of multiple states is a fundamental phenomenon in biology systems and has been the focus of many experimental and computational studies. This work presents a simulation method to directly study the equilibrium of multiple states. This method constructs a virtual mixture of multiple states (VMMS) to sample the conformational space of all chemical states simultaneously. The VMMS system consists of multiple subsystems, one for each state. The subsystem contains a solute and a solvent environment. The solute molecules in all subsystems share the same conformation but have their own solvent environments. Transition between states is implicated by the change of their molar fractions. Simulation of a VMMS system allows efficient calculation of relative free energies of all states, which in turn determine their equilibrium molar fractions. For systems with a large number of state transition sites, an implicit site approximation is introduced to minimize the cost of simulation. A direct application of the VMMS method is for constant pH simulation to study protonation equilibrium. Applying the VMMS method to a heptapeptide of 3 ionizable residues, we calculated the pKas of those residues both with all explicit states and with implicit sites and obtained consistent results. For mouse epidermal growth factor of 9 ionizable groups, our VMMS simulations with implicit sites produced pKas of all 9 ionizable groups and the results agree qualitatively with NMR measurement. This example demonstrates the VMMS method can be applied to systems of a large number of ionizable groups and the computational cost scales linearly with the number of ionizable groups. For one of the most challenging systems in constant pH calculation, SNase Δ+PHS/V66K, our VMMS simulation shows that it is the state-dependent water penetration that causes the large deviation in lysine66’s pKa.

by Daniel A. Keedy, James S. Fraser, Henry van den Bedem

Proteins must move between different conformations of their native ensemble to perform their functions. Crystal structures obtained from high-resolution X-ray diffraction data reflect this heterogeneity as a spatial and temporal conformational average. Although movement between natively populated alternative conformations can be critical for characterizing molecular mechanisms, it is challenging to identify these conformations within electron density maps. Alternative side chain conformations are generally well separated into distinct rotameric conformations, but alternative backbone conformations can overlap at several atomic positions. Our model building program qFit uses mixed integer quadratic programming (MIQP) to evaluate an extremely large number of combinations of sidechain conformers and backbone fragments to locally explain the electron density. Here, we describe two major modeling enhancements to qFit: peptide flips and alternative glycine conformations. We find that peptide flips fall into four stereotypical clusters and are enriched in glycine residues at the n+1 position. The potential for insights uncovered by new peptide flips and glycine conformations is exemplified by HIV protease, where different inhibitors are associated with peptide flips in the “flap” regions adjacent to the inhibitor binding site. Our results paint a picture of peptide flips as conformational switches, often enabled by glycine flexibility, that result in dramatic local rearrangements. Our results furthermore demonstrate the power of large-scale computational analysis to provide new insights into conformational heterogeneity. Overall, improved modeling of backbone heterogeneity with high-resolution X-ray data will connect dynamics to the structure-function relationship and help drive new design strategies for inhibitors of biomedically important systems.

by Manuel Wahle, Willy Wriggers

The modeling of large biomolecular assemblies relies on an efficient rendering of their hierarchical architecture across a wide range of spatial level of detail. We describe a paradigm shift currently under way in computer graphics towards the use of more realistic global illumination models, and we apply the so-called ambient occlusion approach to our open-source multi-scale modeling program, Sculptor. While there are many other higher quality global illumination approaches going all the way up to full GPU-accelerated ray tracing, they do not provide size-specificity of the features they shade. Ambient occlusion is an aspect of global lighting that offers great visual benefits and powerful user customization. By estimating how other molecular shape features affect the reception of light at some surface point, it effectively simulates indirect shadowing. This effect occurs between molecular surfaces that are close to each other, or in pockets such as protein or ligand binding sites. By adding ambient occlusion, large macromolecular systems look much more natural, and the perception of characteristic surface features is strongly enhanced. In this work, we present a real-time implementation of screen space ambient occlusion that delivers realistic cues about tunable spatial scale characteristics of macromolecular architecture. Heretofore, the visualization of large biomolecular systems, comprising e.g. hundreds of thousands of atoms or Mega-Dalton size electron microscopy maps, did not take into account the length scales of interest or the spatial resolution of the data. Our approach has been uniquely customized with shading that is tuned for pockets and cavities of a user-defined size, making it useful for visualizing molecular features at multiple scales of interest. This is a feature that none of the conventional ambient occlusion approaches provide. Actual Sculptor screen shots illustrate how our implementation supports the size-dependent rendering of molecular surface features.

by Sivan Leviyang, Vitaly V. Ganusov

Recent studies have highlighted the ability of HIV to escape from cytotoxic T lymphocyte (CTL) responses that concurrently target multiple viral epitopes. Yet, the viral dynamics involved in such escape are incompletely understood. Previous analyses have made several strong assumptions regarding HIV escape from CTL responses such as independent or non-concurrent escape from individual CTL responses. Using experimental data from evolution of HIV half genomes in four patients we observe concurrent viral escape from multiple CTL responses during early infection (first 100 days of infection), providing confirmation of a recent result found in a study of one HIV-infected patient. We show that current methods of estimating CTL escape rates, based on the assumption of independent escapes, are biased and perform poorly when CTL escape proceeds concurrently at multiple epitopes. We propose a new method for analyzing longitudinal sequence data to estimate the rate of CTL escape across multiple epitopes; this method involves few parameters and performs well in simulation studies. By applying our novel method to experimental data, we find that concurrent multiple escapes occur at rates between 0.03 and 0.4 day−1, a relatively broad range that reflects uncertainty due to sparse sampling and wide ranges of parameter values. However, we show that concurrent escape at rates 0.1–0.2 day−1 across multiple epitopes is consistent with our patient datasets.

by Seren Soner, Pemra Ozbek, Jose Ignacio Garzon, Nir Ben-Tal, Turkan Haliloglu

Protein-protein interfaces have been evolutionarily-designed to enable transduction between the interacting proteins. Thus, we hypothesize that analysis of the dynamics of the complex can reveal details about the nature of the interaction, and in particular whether it is obligatory, i.e., persists throughout the entire lifetime of the proteins, or not. Indeed, normal mode analysis, using the Gaussian network model, shows that for the most part obligatory and non-obligatory complexes differ in their decomposition into dynamic domains, i.e., the mobile elements of the protein complex. The dynamic domains of obligatory complexes often mix segments from the interacting chains, and the hinges between them do not overlap with the interface between the chains. In contrast, in non-obligatory complexes the interface often hinges between dynamic domains, held together through few anchor residues on one side of the interface that interact with their counterpart grooves in the other end. In automatic analysis, 117 of 139 obligatory (84.2%) and 203 of 246 non-obligatory (82.5%) complexes are correctly classified by our method: DynaFace. We further use DynaFace to predict obligatory and non-obligatory interactions among a set of 300 putative protein complexes. DynaFace is available at: http://safir.prc.boun.edu.tr/dynaface.

by Priyanka Prakash, Abdallah Sayyed-Ahmad, Alemayehu A. Gorfe

Probe-based or mixed solvent molecular dynamics simulation is a useful approach for the identification and characterization of druggable sites in drug targets. However, thus far the method has been applied only to soluble proteins. A major reason for this is the potential effect of the probe molecules on membrane structure. We have developed a technique to overcome this limitation that entails modification of force field parameters to reduce a few pairwise non-bonded interactions between selected atoms of the probe molecules and bilayer lipids. We used the resulting technique, termed pMD-membrane, to identify allosteric ligand binding sites on the G12D and G13D oncogenic mutants of the K-Ras protein bound to a negatively charged lipid bilayer. In addition, we show that differences in probe occupancy can be used to quantify changes in the accessibility of druggable sites due to conformational changes induced by membrane binding or mutation.

by Matteo Tiberti, Elena Papaleo, Tone Bengtsen, Wouter Boomsma, Kresten Lindorff-Larsen

There is increasing evidence that protein dynamics and conformational changes can play an important role in modulating biological function. As a result, experimental and computational methods are being developed, often synergistically, to study the dynamical heterogeneity of a protein or other macromolecules in solution. Thus, methods such as molecular dynamics simulations or ensemble refinement approaches have provided conformational ensembles that can be used to understand protein function and biophysics. These developments have in turn created a need for algorithms and software that can be used to compare structural ensembles in the same way as the root-mean-square-deviation is often used to compare static structures. Although a few such approaches have been proposed, these can be difficult to implement efficiently, hindering a broader applications and further developments. Here, we present an easily accessible software toolkit, called ENCORE, which can be used to compare conformational ensembles generated either from simulations alone or synergistically with experiments. ENCORE implements three previously described methods for ensemble comparison, that each can be used to quantify the similarity between conformational ensembles by estimating the overlap between the probability distributions that underlie them. We demonstrate the kinds of insights that can be obtained by providing examples of three typical use-cases: comparing ensembles generated with different molecular force fields, assessing convergence in molecular simulations, and calculating differences and similarities in structural ensembles refined with various sources of experimental data. We also demonstrate efficient computational scaling for typical analyses, and robustness against both the size and sampling of the ensembles. ENCORE is freely available and extendable, integrates with the established MDAnalysis software package, reads ensemble data in many common formats, and can work with large trajectory files.

by Sedeer el-Showk, Hanna Help-Rinta-Rahko, Tiina Blomster, Riccardo Siligato, Athanasius F. M. Marée, Ari Pekka Mähönen, Verônica A. Grieneisen

An auxin maximum is positioned along the xylem axis of the Arabidopsis root tip. The pattern depends on mutual feedback between auxin and cytokinins mediated by the PIN class of auxin efflux transporters and AHP6, an inhibitor of cytokinin signalling. This interaction has been proposed to regulate the size and the position of the hormones’ respective signalling domains and specify distinct boundaries between them. To understand the dynamics of this regulatory network, we implemented a parsimonious computational model of auxin transport that considers hormonal regulation of the auxin transporters within a spatial context, explicitly taking into account cell shape and polarity and the presence of cell walls. Our analysis reveals that an informative spatial pattern in cytokinin levels generated by diffusion is a theoretically unlikely scenario. Furthermore, our model shows that such a pattern is not required for correct and robust auxin patterning. Instead, auxin-dependent modifications of cytokinin response, rather than variations in cytokinin levels, allow for the necessary feedbacks, which can amplify and stabilise the auxin maximum. Our simulations demonstrate the importance of hormonal regulation of auxin efflux for pattern robustness. While involvement of the PIN proteins in vascular patterning is well established, we predict and experimentally verify a role of AUX1 and LAX1/2 auxin influx transporters in this process. Furthermore, we show that polar localisation of PIN1 generates an auxin flux circuit that not only stabilises the accumulation of auxin within the xylem axis, but also provides a mechanism for auxin to accumulate specifically in the xylem-pole pericycle cells, an important early step in lateral root initiation. The model also revealed that pericycle cells on opposite xylem poles compete for auxin accumulation, consistent with the observation that lateral roots are not initiated opposite to each other.

by Lane W. Votapka, Rommie E. Amaro

The kinetic rate constants of binding were estimated for four biochemically relevant molecular systems by a method that uses milestoning theory to combine Brownian dynamics simulations with more detailed molecular dynamics simulations. The rate constants found using this method agreed well with experimentally and theoretically obtained values. We predicted the association rate of a small charged molecule toward both a charged and an uncharged spherical receptor and verified the estimated value with Smoluchowski theory. We also calculated the kon rate constant for superoxide dismutase with its natural substrate, O2−, in a validation of a previous experiment using similar methods but with a number of important improvements. We also calculated the kon for a new system: the N-terminal domain of Troponin C with its natural substrate Ca2+. The kon calculated for the latter two systems closely resemble experimentally obtained values. This novel multiscale approach is computationally cheaper and more parallelizable when compared to other methods of similar accuracy. We anticipate that this methodology will be useful for predicting kinetic rate constants and for understanding the process of binding between a small molecule and a protein receptor.

by M. Olivia Kim, Patrick G. Blachly, J. Andrew McCammon

BACE-1 is the β-secretase responsible for the initial amyloidogenesis in Alzheimer’s disease, catalyzing hydrolytic cleavage of substrate in a pH-sensitive manner. The catalytic mechanism of BACE-1 requires water-mediated proton transfer from aspartyl dyad to the substrate, as well as structural flexibility in the flap region. Thus, the coupling of protonation and conformational equilibria is essential to a full in silico characterization of BACE-1. In this work, we perform constant pH replica exchange molecular dynamics simulations on both apo BACE-1 and five BACE-1-inhibitor complexes to examine the effect of pH on dynamics and inhibitor binding properties of BACE-1. In our simulations, we find that solution pH controls the conformational flexibility of apo BACE-1, whereas bound inhibitors largely limit the motions of the holo enzyme at all levels of pH. The microscopic pKa values of titratable residues in BACE-1 including its aspartyl dyad are computed and compared between apo and inhibitor-bound states. Changes in protonation between the apo and holo forms suggest a thermodynamic linkage between binding of inhibitors and protons localized at the dyad. Utilizing our recently developed computational protocol applying the binding polynomial formalism to the constant pH molecular dynamics (CpHMD) framework, we are able to obtain the pH-dependent binding free energy profiles for various BACE-1-inhibitor complexes. Our results highlight the importance of correctly addressing the binding-induced protonation changes in protein-ligand systems where binding accompanies a net proton transfer. This work comprises the first application of our CpHMD-based free energy computational method to protein-ligand complexes and illustrates the value of CpHMD as an all-purpose tool for obtaining pH-dependent dynamics and binding free energies of biological systems.

by Benfeard Williams II, Marino Convertino, Jhuma Das, Nikolay V. Dokholyan

The increased risk of developing Alzheimer’s disease (AD) is associated with the APOE gene, which encodes for three variants of Apolipoprotein E, namely E2, E3, E4, differing only by two amino acids at positions 112 and 158. ApoE4 is known to be the strongest risk factor for AD onset, while ApoE3 and ApoE2 are considered to be the AD-neutral and AD-protective isoforms, respectively. It has been hypothesized that the ApoE isoforms may contribute to the development of AD by modifying the homeostasis of ApoE physiological partners and AD-related proteins in an isoform-specific fashion. Here we find that, despite the high sequence similarity among the three ApoE variants, only ApoE4 exhibits a misfolded intermediate state characterized by isoform-specific domain-domain interactions in molecular dynamics simulations. The existence of an ApoE4-specific intermediate state can contribute to the onset of AD by altering multiple cellular pathways involved in ApoE-dependent lipid transport efficiency or in AD-related protein aggregation and clearance. We present what we believe to be the first structural model of an ApoE4 misfolded intermediate state, which may serve to elucidate the molecular mechanism underlying the role of ApoE4 in AD pathogenesis. The knowledge of the structure for the ApoE4 folding intermediate provides a new platform for the rational design of alternative therapeutic strategies to fight AD.

by Rong Shen, Wei Han, Giacomo Fiorin, Shahidul M. Islam, Klaus Schulten, Benoît Roux

The knowledge of multiple conformational states is a prerequisite to understand the function of membrane transport proteins. Unfortunately, the determination of detailed atomic structures for all these functionally important conformational states with conventional high-resolution approaches is often difficult and unsuccessful. In some cases, biophysical and biochemical approaches can provide important complementary structural information that can be exploited with the help of advanced computational methods to derive structural models of specific conformational states. In particular, functional and spectroscopic measurements in combination with site-directed mutations constitute one important source of information to obtain these mixed-resolution structural models. A very common problem with this strategy, however, is the difficulty to simultaneously integrate all the information from multiple independent experiments involving different mutations or chemical labels to derive a unique structural model consistent with the data. To resolve this issue, a novel restrained molecular dynamics structural refinement method is developed to simultaneously incorporate multiple experimentally determined constraints (e.g., engineered metal bridges or spin-labels), each treated as an individual molecular fragment with all atomic details. The internal structure of each of the molecular fragments is treated realistically, while there is no interaction between different molecular fragments to avoid unphysical steric clashes. The information from all the molecular fragments is exploited simultaneously to constrain the backbone to refine a three-dimensional model of the conformational state of the protein. The method is illustrated by refining the structure of the voltage-sensing domain (VSD) of the Kv1.2 potassium channel in the resting state and by exploring the distance histograms between spin-labels attached to T4 lysozyme. The resulting VSD structures are in good agreement with the consensus model of the resting state VSD and the spin-spin distance histograms from ESR/DEER experiments on T4 lysozyme are accurately reproduced.