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Structures of biomolecular systems are increasingly computed by integrative modeling that relies on varied types of experimental data and theoretical information. We describe here the proceedings and conclusions from the first wwPDB Hybrid/Integrative Methods Task Force Workshop held at the European Bioinformatics Institute in Hinxton, UK, on October 6 and 7, 2014. At the workshop, experts in various experimental fields of structural biology, experts in integrative modeling and visualization, and experts in data archiving addressed a series of questions central to the future of structural biology. How should integrative models be represented? How should the data and integrative models be validated? What data should be archived? How should the data and models be archived? What information should accompany the publication of integrative models?, (Copyright © 2015 Elsevier Ltd. All rights reserved.)

BamA is the main component of the β-barrel assembly machinery (BAM) that folds and inserts outer membrane proteins in Gram-negative bacteria. Crystal structures have suggested that this process involves conformational changes in the transmembrane β-barrel of BamA that allow for lateral opening, as well as large overall rearrangements of its periplasmic POTRA domains. Here, we identify local dynamics of the BamA POTRA 5 domain by solution and solid-state nuclear magnetic resonance. The protein region undergoing conformational exchange is highly conserved and contains residues critical for interaction with BamD and correct β-barrel assembly in vivo. We show that mutations known to affect the latter processes influence the conformational equilibrium, suggesting that the plasticity of POTRA 5 is related to its interaction with BamD and possibly to substrate binding. Taken together, a view emerges in which local protein plasticity may be critically involved in the different stages of outer membrane protein folding and insertion., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The integration of biophysical data from multiple sources is critical for developing accurate structural models of large multiprotein systems and their regulators. Mass spectrometry (MS) can be used to measure the insertion location for a wide range of topographically sensitive chemical probes, and such insertion data provide a rich, but disparate set of modeling restraints. We have developed a software platform that integrates the analysis of label-based MS and tandem MS (MS(2)) data with protein modeling activities (Mass Spec Studio). Analysis packages can mine any labeling data from any mass spectrometer in a proteomics-grade manner, and link labeling methods with data-directed protein interaction modeling using HADDOCK. Support is provided for hydrogen/deuterium exchange (HX) and covalent labeling chemistries, including novel acquisition strategies such as targeted HX-MS(2) and data-independent HX-MS(2). The latter permits the modeling of highly complex systems, which we demonstrate by the analysis of microtubule interactions., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Elemental biological functions such as molecular signal transduction are determined by the dynamic interplay between polypeptides and the membrane environment. Determining such supramolecular arrangements poses a significant challenge for classical structural biology methods. We introduce an iterative approach that combines magic-angle spinning solid-state NMR spectroscopy and atomistic molecular dynamics simulations for the determination of the structure and topology of membrane-bound systems with a resolution and level of accuracy difficult to obtain by either method alone. Our study focuses on the Shaker B ball peptide that is representative for rapid N-type inactivating domains of voltage-gated K(+) channels, associated with negatively charged lipid bilayers., (Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The protocols currently used for protein structure determination by nuclear magnetic resonance (NMR) depend on the determination of a large number of upper distance limits for proton-proton pairs. Typically, this task is performed manually by an experienced researcher rather than automatically by using a specific computer program. To assess whether it is indeed possible to generate in a fully automated manner NMR structures adequate for deposition in the Protein Data Bank, we gathered 10 experimental data sets with unassigned nuclear Overhauser effect spectroscopy (NOESY) peak lists for various proteins of unknown structure, computed structures for each of them using different, fully automatic programs, and compared the results to each other and to the manually solved reference structures that were not available at the time the data were provided. This constitutes a stringent "blind" assessment similar to the CASP and CAPRI initiatives. This study demonstrates the feasibility of routine, fully automated protein structure determination by NMR., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Binding-induced backbone and large-scale conformational changes represent one of the major challenges in the modeling of biomolecular complexes by docking. To address this challenge, we have developed a flexible multidomain docking protocol that follows a "divide-and-conquer" approach to model both large-scale domain motions and small- to medium-scale interfacial rearrangements: the flexible binding partner is treated as an assembly of subparts/domains that are docked simultaneously making use of HADDOCK's multidomain docking ability. For this, the flexible molecules are cut at hinge regions predicted using an elastic network model. The performance of this approach is demonstrated on a benchmark covering an unprecedented range of conformational changes of 1.5 to 19.5 Å. We show from a statistical survey of known complexes that the cumulative sum of eigenvalues obtained from the elastic network has some predictive power to indicate the extent of the conformational change to be expected., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Recognition of signal sequences by cognate receptors controls the entry of virtually all proteins to export pathways. Despite its importance, this process remains poorly understood. Here, we present the solution structure of a signal peptide bound to SecA, the 204 kDa ATPase motor of the Sec translocase. Upon encounter, the signal peptide forms an alpha-helix that inserts into a flexible and elongated groove in SecA. The mode of binding is bimodal, with both hydrophobic and electrostatic interactions mediating recognition. The same groove is used by SecA to recognize a diverse set of signal sequences. Impairment of the signal-peptide binding to SecA results in significant translocation defects. The C-terminal tail of SecA occludes the groove and inhibits signal-peptide binding, but autoinhibition is relieved by the SecB chaperone. Finally, it is shown that SecA interconverts between two conformations in solution, suggesting a simple mechanism for polypeptide translocation.

We report an experimental and theoretical study on type VIII beta-turn using a designed peptide of sequence GDNP. CD and NMR studies reveal that this peptide exists in equilibrium between type VIII beta-turn and extended conformations. Extensive MD simulations give a description of the free energy landscape of the peptide in which we retrieve the same two main conformations suggested by the experiments. The free energy difference between the two conformational states is very small and the transition between them occurs within a few kT at 300 K on a nanosecond timescale. The equilibrium is mainly driven by entropic contribution, which favors extended conformations over beta-turns. This confirms other theoretical studies showing that beta-turns are marginally stable in water solution because of the larger entropy of the extended state unless some stabilizing interactions exist. Our observations may be extended to any type of beta-turn and have important consequences for protein folding. A comparison of our MD and CD results also suggests a possible type VIII beta-turn CD signature indicated by one main band at 200 nm, close to that of random coil, and a fairly large shoulder at 220 nm. Last, our results clearly show that the XXXP motif can only fold into a type VIII beta-turn, which is consistent with its fairly strong propensity for this type of turn. This important finding may help for peptide design and is in line with recent studies on bioactive elastin peptides.

The HIV-1 gp120/CD4 interaction shows a large, unprecedented entropy/enthalpy compensation, with the capacity to fine-tune recognition over a broad range of affinity. The intermolecular interaction involves stable hydrophobic contacts with a unique protruding CD4-Phe43 structure surrounded by an intermolecular hydrogen-bond network that covers the hemisphere of the CD4 D1 domain. We have applied a heuristic formula based on the covariance matrix of atom-positional fluctuations to assess the configurational entropy of the gp120/CD4 complex at different levels. The system was dissected into various subsets of atoms to evaluate the entropic contributions of different functional elements. By combining the trajectories of the free and complex forms, further insight into the conformational sampling was extracted. Despite the limited sampling time of 10 ns, the theoretically derived changes in configurational entropy are in fair agreement with the experimentally determined data. The simultaneous evaluation of different interaction modes through a decomposition approach is only feasible with the knowledge of the atomic trajectory of the system. The configurational entropy analysis in terms of combined trajectories presented here shall potentially provide accurate estimations of thermodynamic properties of biomolecules given sufficient sampling of conformational space.

The protein CNOT4 possesses an N-terminal RING finger domain that acts as an E3 ubiquitin ligase and specifically interacts with UbcH5B, a ubiquitin-conjugating enzyme. The structure of the CNOT4 RING domain has been solved and the amino acids important for the binding to UbcH5B have been mapped. Here, the residues of UbcH5B important for the binding to CNOT4 RING domain were identified by NMR chemical shift perturbation experiments, and these data were used to generate structural models of the complex with the program HADDOCK. Together with the NMR data, additional biochemical data were included in a second docking, and comparisons of the resulting model with the structure of the c-Cbl/UbcH7 complex reveal some significant differences, notably at specific residues, and give structural insights into the E2/E3 specificity.

A structural model of the transient complex between the yeast copper chaperone Atx1 and the first soluble domain of the copper transporting ATPase Ccc2 was obtained with HADDOCK, combining NMR chemical shift mapping information with in silico docking. These two proteins are involved in copper trafficking in yeast cells. Calculations were performed starting with the copper ion either bound to Atx1 or to Ccc2 and using the experimental structures of the copper-loaded and apo forms of each protein. The copper binding motifs of the two proteins are found in close proximity. Copper tends to move from Atx1 to Ccc2, consistent with the physiological direction of transfer, with concomitant structural rearrangements, in agreement with experimental observations. The interaction is mainly of an electrostatic nature with hydrogen bonds stabilizing the complex. The structural data are relevant for a number of proteins homologous to Atx1 and Ccc2 and conserved from bacteria to humans.

Background: Lactose repressor protein (Lac) controls the expression of the lactose metabolic genes in Escherichia coli by binding to an operator sequence in the promoter of the lac operon. Binding of inducer molecules to the Lac core domain induces changes in tertiary structure that are propagated to the DNA-binding domain through the connecting hinge region, thereby reducing the affinity for the operator. Protein-protein and protein-DNA interactions involving the hinge region play a crucial role in the allosteric changes occurring upon induction, but have not, as yet, been analyzed in atomic detail., Results: We have used nuclear magnetic resonance (NMR) spectroscopy and restrained molecular dynamics (rMD) to determine the structure of the Lac repressor DNA-binding domain (headpeice 62; HP62) in complex with a symmetrized lac operator. Analysis of the structures reveals specific interactions between Lac repressor and DNA that were not found in previously investigated Lac repressor-DNA complexes. Important differences with the previously reported structures of the HP56-DNA complex were found in the loop following the helix-turn-helix (HTH) motif. The protein-protein and protein-DNA interactions involving the hinge region and the deformations in the DNA structure could be delineated in atomic detail. The structures were also used for comparison with the available crystallographic data on the Lac and Pur repressor-DNA complexes., Conclusions: The structures of the HP62-DNA complex provide the basis for a better understanding of the specific recognition in the Lac repressor-operator complex. In addition, the structural features of the hinge region provide detailed insight into the protein-protein and protein-DNA interactions responsible for the high affinity of the repressor for operator DNA.