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Recent technological advances in NMR spectroscopy have alleviated the size limitations for the determination of biomolecular structures in solution. At the same time, novel NMR parameters such as residual dipolar couplings are providing greater accuracy. As this review shows, the structures of protein-protein and protein-nucleic acid complexes up to 50 kDa can now be accurately determined. Although de novo structure determination still requires considerable effort, information on interaction surfaces from chemical shift perturbations is much easier to obtain. Advances in modelling and data-driven docking procedures allow this information to be used for determining approximate structures of biomolecular complexes. As a result, a wealth of information has become available on the way in which proteins interact with other biomolecules. Of particular interest is the fact that these NMR-based methods can be applied to weak and transient protein-protein complexes that are difficult to study by other structural methods.

We have shown previously that given high-resolution structures of the unbound molecules, structure determination of protein com- plexes is possible by including biochemical and/or biophysical data as highly ambiguous distance re- straints in a docking approach. We applied this method, implemented in the HADDOCK (High Ambi- guity Driven DOCKing) package (Dominguez et al., J Am Chem Soc 2003;125:1731-1737), to the targets in the fourth and fifth rounds of CAPRI. Here we describe our results and analyze them in detail. Special attention is given to the role of flexibility in our docking method and the way in which this improves the docking results. We describe exten- sions to our approach that were developed as a direct result of our participation in CAPRI. In addi- tion to experimental information, we also included interface residue predictions from PPISP (Protein- Protein Interaction Site Predictor; Zhou and Shan, Proteins 2001;44:336 -343), a neural network method. Using HADDOCK we were able to generate accept- able structures for 6 of the 8 targets, and to submit at least 1 acceptable structure for 5 of them. Of these 5 submissions, 3 were of medium quality (Targets 10, 11, and 15) and 2 of high quality (Targets 13 and 14). In all cases, predictions were obtained containing at least 40% of the correct epitope at the interface for both ligand and receptor simultaneously. Proteins 2005;60:232-238. © 2005 Wiley-Liss, Inc.

We present two new databases of NMR-derived distance and dihedral angle restraints: the Database Of Converted Restraints (DOCR) and the Filtered Restraints Database (FRED). These databases currently correspond to 545 proteins with NMR structures deposited in the Protein Databank (PDB). The criteria for inclusion were that these should be unique, monomeric proteins with author-provided experimental NMR data and coordinates available from the PDB capable of being parsed and prepared in a consistent manner. The Wattos program was used to parse the files, and the CcpNmr FormatConverter program was used to prepare them semi-automatically. New modules, including a new implementation of Aqua in the BioMagResBank (BMRB) software Wattos were used to analyze the sets of distance restraints (DRs) for inconsistencies, redundancies, NOE completeness, classification and violations with respect to the original coordinates. Restraints that could not be associated with a known nomenclature were flagged. The coordinates of hydrogen atoms were recalculated from the positions of heavy atoms to allow for a full restraint analysis. The DOCR database contains restraint and coordinate data that is made consistent with each other and with IUPAC conventions. The FRED database is based on the DOCR data but is filtered for use by test calculation protocols and longitudinal analyses and validations. These two databases are available from websites of the BMRB and the Macromolecular Structure Database (MSD) in various formats: NMR-STAR, CCPN XML, and in formats suitable for direct use in the software packages CNS and CYANA.

A 0.2 μs molecular dynamics simulation of ubiquitin in water is presented, which allows us to assess both the global tumbling in solution and the internal dynamics. The latter reveals slow motions outside the classical NMR timewindow, in agreement with recent RDC and cross-correlation measurements. Analysis of back-calculated relaxation rates using the classical NMR model-free approach reproduces the amplitudes of internal motions expressed in the order parameter, while it severely underestimates the corresponding time scales present in the simulation.

Proteins involved in signal transduction can usually be present in two states: an inactive and an active (signaling) state. In the case of photoreceptors such as PYP, it has been shown that the signaling state has a large degree of structural and dynamic disorder. Conventional structural NMR approaches present difficulties in describing such partially unfolded states. Owing to the disordered dynamical and transient nature of such states classical NOE-based information, when present, is sparse. Chemical shift changes upon partial unfolding can, however, be easily monitored from HSQC spectra. We show here that such states can be modeled by defining native-like inter-residue contacts for those residues that do not shift significantly upon partial unfolding. The feasibility of this approach is demonstrated using lysozyme as a test case and applied to model the partially unfolded signaling state (pB) of a truncated form of the photoactive yellow protein for which a ‘‘classical’’ NOE-based structure is available for validation. This approach should be generally applicable to systems in which part of the structure remains in a well-defined native-like conformation.

State-of-the-art methods based on CNS and CYANA were used to recalculate the nuclear magnetic resonance (NMR) solution structures of 500 proteins for which coordinates and NMR restraints are available from the Protein Data Bank. Curated restraints were obtained from the BioMagResBank FRED database. Although the original NMR struc- tures were determined by various methods, they all were recalculated by CNS and CYANA and refined subsequently by restrained molecular dynamics (CNS) in a hydrated environment. We present an extensive analysis of the results, in terms of various quality indicators generated by PROCHECK and WHAT- _CHECK. On average, the quality indicators for pack- ing and Ramachandran appearance moved one stan- dard deviation closer to the mean of the reference database. The structural quality of the recalculated structures is discussed in relation to various parame- ters, including number of restraints per residue, NOE completeness and positional root mean square devia- tion (RMSD). Correlations between pairs of these quality indicators were generally low; for example, there is a weak correlation between the number of restraints per residue and the Ramachandran appear- ance according to WHAT_CHECK (r 0.31). The set of recalculated coordinates constitutes a unified data- base of protein structures in which potential user- and software-dependent biases have been kept as small as possible. The database can be used by the structural biology community for further develop- ment of calculation protocols, validation tools, struc- ture-based statistical approaches and modeling. The RECOORD database of recalculated structures is pub- licly available from http://www.ebi.ac.uk/msd/reco

The interaction of bovine microsomal ferricytochrome b5 with yeast iso-1-ferri and ferrocytochrome c has been investigated using heteronuclear NMR techniques. Chemical-shift perturbations for 1H and 15N nuclei of both cytochromes, arising from the interactions with the unlabeled partner proteins, were used for mapping the interacting surfaces on both proteins. The similarity of the binding shifts observed for oxidized and reduced cytochrome c indicates that the complex formation is not influenced by the oxidation state of the cytochrome c. Protein–protein docking simulations have been performed for the binary cytochrome b5–cytochrome c and ternary (cytochrome b5)–(cytochrome c)2 complexes using a novel HADDOCK approach. The docking procedure, which makes use of the experimental data to drive the docking, identified a range of orientations assumed by the proteins in the complex. It is demonstrated that cytochrome c uses a confined surface patch for interaction with a much more extensive surface area of cytochrome b5. Taken together, the experimental data suggest the presence of a dynamic ensemble of conformations assumed by the proteins in the complex.

Sequence-specific high mobility group (HMG) box factors bind and bend DNA via interactions in the minor groove. Three-dimensional NMR analyses have provided the structural basis for this interaction. The cognate HMG domain DNA motif is generally believed to span 6-8 bases. However, alignment of promoter elements controlled by the yeast genes ste11 and Rox1 has indicated strict conservation of a larger DNA motif. By site selection, we identify a highly specific 12-base pair motif for Ste11, AGAACAAAGAAA. Similarly, we show that Tcf1, MatMc, and Sox4 bind unique, highly specific DNA motifs of 12, 12, and 10 base pairs, respectively. Footprinting with a deletion mutant of Ste11 reveals a novel interaction between the 3' base pairs of the extended DNA motif and amino acids C-terminal to the HMG domain. The sequence-specific interaction of Ste11 with these 3' base pairs contributes significantly to binding and bending of the DNA motif.

When classical, Nuclear Overhauser Effect (NOE)-based approaches fail, it is possible, given high-resolution structures of the free molecules, to model the structure of a complex in solution based solely on chemical shift perturbation (CSP) data in combination with orientational restraints from residual dipolar couplings (RDCs) when available. RDCs can be incorporated into the docking following various strategies: as direct restraints and/or as intermolecular intervector projection angle restraints (Meiler et al., J Biomol NMR 2000;16:245-252). The advantage of the latter for docking is that they directly define the relative orientation of the molecules. A combined protocol in which RDCs are first introduced as intervector projection angle restraints and at a later stage as direct restraints is shown here to give the best performance. This approach, implemented in our information-driven docking approach HADDOCK (Dominguez et al., J Am Chem Soc 2003;125:1731-1737), is used to determine the solution structure of the Lys48-linked di-ubiquitin, for which chemical shift mapping, RDCs, and (15)N-relaxation data have been previously obtained (Varadan et al., J Mol Biol 2002;324:637-647). The resulting structures, derived from CSP and RDC data, are cross-validated using (15)N-relaxation data. The solution structure differs from the crystal structure by a 20 degrees rotation of the two ubiquitin units relative to each other.

Herpes simplex virion protein 16 (VP16) contains two strong activation regions that can independently and cooperatively activate transcription in vivo. We have identified the regions and residues involved in the interaction with the human transcriptional coactivator positive cofactor 4 (PC4) and the general transcription factor TFIIB. NMR and biochemical experiments revealed that both VP16 activation regions are required for the interaction and undergo a conformational transition from random coil to alpha-helix upon binding to its target PC4. The interaction is strongly electrostatically driven and the binding to PC4 is enhanced by the presence of its amino-terminal domain. We propose models for binding of VP16 to the core domains of PC4 and TFIIB that are based on two independent docking approaches using NMR chemical shift changes observed in titration experiments. The models are consistent with results from site-directed mutagenesis and provide an explanation for the contribution of both acidic and hydrophobic residues for transcriptional activation by VP16. Both intrinsically unstructured activation domains are attracted to their interaction partner by electrostatic interactions, and adopt an alpha-helical conformation around the important hydrophobic residues. The models showed multiple distinct binding surfaces upon interaction with various partners, providing an explanation for the promiscuous properties, cooperativity, and the high activity of this activation domain.

SummaryThe human ERCC1/XPF complex is a structure-specific endonuclease with defined polarity that participates in multiple DNA repair pathways. We report the heterodimeric structure of the C-terminal domains of both proteins responsible for ERCC1/XPF complex formation. Both domains exhibit the double helix-hairpin-helix motif (HhH)2, and they are related by a pseudo-2-fold symmetry axis. In the XPF domain, the hairpin of the second motif is replaced by a short turn. The ERCC1 domain folds properly only in the presence of the XPF domain, which implies a role for XPF as a scaffold for the folding of ERCC1. The intersubunit interactions are largely hydrophobic in nature. NMR titration data show that only the ERCC1 domain of the ERCC1/XPF complex is involved in DNA binding. On the basis of these findings, we propose a model for the targeting of XPF nuclease via ERCC1-mediated interactions in the context of nucleotide excision repair.

Sequence-specific high mobility group (HMG) box factors bind and bend DNA via interactions in the minor groove. Three-dimensional NMR analyses have provided the structural basis for this interaction. The cognate HMG domain DNA motif is generally believed to span 6-8 bases. However, alignment of promoter elements controlled by the yeast genes ste11 and Rox1 has indicated strict conservation of a larger DNA motif. By site selection, we identify a highly specific 12-base pair motif for Ste11, AGAACAAAGAAA. Similarly, we show that Tcf1, MatMc, and Sox4 bind unique, highly specific DNA motifs of 12, 12, and 10 base pairs, respectively. Footprinting with a deletion mutant of Ste11 reveals a novel interaction between the 3' base pairs of the extended DNA motif and amino acids C-terminal to the HMG domain. The sequence-specific interaction of Ste11 with these 3' base pairs contributes significantly to binding and bending of the DNA motif.

The interaction of bovine microsomal ferricytochrome b5 with yeast iso-1-ferri and ferrocytochrome c has been investigated using heteronuclear NMR techniques. Chemical-shift perturbations for 1H and 15N nuclei of both cytochromes, arising from the interactions with the unlabeled partner proteins, were used for mapping the interacting surfaces on both proteins. The similarity of the binding shifts observed for oxidized and reduced cytochrome c indicates that the complex formation is not influenced by the oxidation state of the cytochrome c. Protein–protein docking simulations have been performed for the binary cytochrome b5–cytochrome c and ternary (cytochrome b5)–(cytochrome c)2 complexes using a novel HADDOCK approach. The docking procedure, which makes use of the experimental data to drive the docking, identified a range of orientations assumed by the proteins in the complex. It is demonstrated that cytochrome c uses a confined surface patch for interaction with a much more extensive surface area of cytochrome b5. Taken together, the experimental data suggest the presence of a dynamic ensemble of conformations assumed by the proteins in the complex.

A 0.2 mu s molecular dynamics simulation of ubiquitin in water is presented, which allows us to assess both the global tumbling in solution and the internal dynamics. The latter reveals slow motions outside the classical NMR timewindow, in agreement with recent RDC and cross-correlation measurements. Analysis of back-calculated relaxation rates using the classical NMR model-free approach reproduces the amplitudes of internal motions expressed in the order parameter, while it severely underestimates the corresponding time scales present in the simulation.

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-Phe(43) 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.

Herpes simplex virion protein 16 (VP16) contains two strong activation regions that can independently and cooperatively activate transcription in vivo. We have identified the regions and residues involved in the interaction with the human transcriptional coactivator positive cofactor 4 (PC4) and the general transcription factor TFIIB. NMR and biochemical experiments revealed that both VP16 activation regions are required for the interaction and undergo a conformational transition from random coil to α-helix upon binding to its target PC4. The interaction is strongly electrostatically driven and the binding to PC4 is enhanced by the presence of its amino-terminal domain. We propose models for binding of VP16 to the core domains of PC4 and TFIIB that are based on two independent docking approaches using NMR chemical shift changes observed in titration experiments. The models are consistent with results from site-directed mutagenesis and provide an explanation for the contribution of both acidic and hydrophobic residues for transcriptional activation by VP16. Both intrinsically unstructured activation domains are attracted to their interaction partner by electrostatic interactions, and adopt an α-helical conformation around the important hydrophobic residues. The models showed multiple distinct binding surfaces upon interaction with various partners, providing an explanation for the promiscuous properties, cooperativity, and the high activity of this activation domain.

International audience; The human general transcription factor TFIIH is involved in both transcription and DNA nucleotide excision repair. Among the 10 subunits of the complex, p44 subunit plays a crucial role in both mechanisms. Its N-terminal domain interacts with the XPD helicase, whereas its C-terminal domain is involved specifically in the promoter escape activity. By mutating an exposed and non-conserved cysteine residue into a serine, we produced a soluble mutant of p44-(321-395) suitable for solution structure determination. The domain adopts a C4C4 RING domain structure with sequential organization of beta-strands that is related to canonical RING domains by a circular permutation of the beta-sheet elements. Analysis of the molecular surface and mutagenesis experiments suggests that the binding of p44-(321-395) to TFIIH p34 subunit is not mediated by electrostatic interactions and, thus, differs from previously reported interaction mechanisms involving RING domains.

State‐of‐the‐art methods based on CNS and CYANA were used to recalculate the nuclear magnetic resonance (NMR) solution structures of 500+ proteins for which coordinates and NMR restraints are available from the Protein Data Bank. Curated restraints were obtained from the BioMagResBank FRED database. Although the original NMR structures were determined by various methods, they all were recalculated by CNS and CYANA and refined subsequently by restrained molecular dynamics (CNS) in a hydrated environment. We present an extensive analysis of the results, in terms of various quality indicators generated by PROCHECK and WHAT_CHECK. On average, the quality indicators for packing and Ramachandran appearance moved one standard deviation closer to the mean of the reference database. The structural quality of the recalculated structures is discussed in relation to various parameters, including number of restraints per residue, NOE completeness and positional root mean square deviation (RMSD). Correlations between pairs of these quality indicators were generally low; for example, there is a weak correlation between the number of restraints per residue and the Ramachandran appearance according to WHAT_CHECK (r = 0.31). The set of recalculated coordinates constitutes a unified database of protein structures in which potential user‐ and software‐dependent biases have been kept as small as possible. The database can be used by the structural biology community for further development of calculation protocols, validation tools, structure‐based statistical approaches and modeling. The RECOORD database of recalculated structures is publicly available from http://www.ebi.ac.uk/msd/recoord. Proteins 2005.