Robert Kaptein

AQUA and PROCHECK-NMR: Programs for checking the quality of protein structures solved by NMR*

SummaryThe AQUA and PROCHECK-NMR programs provide a means of validating the geometry and restraint violations of an ensemble of protein structures solved by solution NMR. The outputs include a detailed breakdown of the restraint violations, a number of plots in PostScript format and summary statistics. These various analyses indicate both the degree of agreement of the model structures with the experimental data, and the quality of their geometrical properties. They are intended to be of use both to support ongoing NMR structure determination and in the validation of the final results.

A protein structure from nuclear magnetic resonance data. lac repressor headpiece.

A procedure is described to determine the three-dimensional structure of biomolecules from nuclear magnetic resonance data. This procedure combines model building with a restrained molecular dynamics algorithm, in which distance information from nuclear Overhauser effects is incorporated in the form of pseudo potentials. The method has been applied to the N-terminal DNA-binding domain or headpiece (amino acid residues 1 to 51) of the lac repressor from Escherichia coli, for which no crystal structure is available. The relative orientation of the three helices of the headpiece is similar to that of the three homologous helices found in the cI repressor of bacteriophage lambda.

The Nisin-Lipid II Complex Reveals a Pyrophosphate Cage that Provides a Blueprint for Novel Antibiotics

The emerging antibiotics-resistance problem has underlined the urgent need for novel antimicrobial agents. Lantibiotics (lanthionine-containing antibiotics) are promising candidates to alleviate this problem. Nisin, a member of this family, has a unique pore-forming activity against bacteria. It binds to lipid II, the essential precursor of cell wall synthesis. As a result, the membrane permeabilization activity of nisin is increased by three orders of magnitude. Here we report the solution structure of the complex of nisin and lipid II. The structure shows a novel lipid II–binding motif in which the pyrophosphate moiety of lipid II is primarily coordinated by the N-terminal backbone amides of nisin via intermolecular hydrogen bonds. This cage structure provides a rationale for the conservation of the lanthionine rings among several lipid II–binding lantibiotics. The structure of the pyrophosphate cage offers a template for structure-based design of novel antibiotics.

RECOORD: A Recalculated Coordinate Database of 500 Proteins from the PDB Using Restraints from the BioMagResBank

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.

Recommendations for the presentation of NMR structures of proteins and nucleic acids – IUPAC-IUBMB-IUPAB Inter-Union Task Group on the Standardization of Data Bases of Protein and Nucleic Acid Structures Determined by NMR Spectroscopy

The recommendations presented here are designed to support easier communication of NMR data and NMR structures of proteins and nucleic acids through unified nomenclature and reporting standards. Much of this document pertains to the reporting of data in journal articles; however, in the interest of the future development of structural biology, it is desirable that the bulk of the reported information be stored in computer-accessible form and be freely accessible to the scientific community in standardized formats for data exchange. These recommendations stem from an IUPAC-IUBMB-IUPAB inter-union venture with the direct involvement of ICSU and CODATA. The Task Group has reviewed previous formal recommendations and has extended them in the light of more recent developments in the field of biomolecular NMR spectroscopy. Drafts of the recommendations presented here have been examined critically by more than 50 specialists in the field and have gone through two rounds of extensive modification to incorporate suggestions and criticisms.

The DNA-binding domain of HIV-1 integrase has an SH3-like fold

We have determined the solution structure of the DNA-binding domain of HIV-1 integrase by nuclear magnetic resonance spectroscopy. In solution, this carboxy-terminal region of integrase forms a homodimer, consisting of two structures that closely resemble Src-homology 3 (SH3) domains. Lys 264, previously identified by mutagenesis studies to be important for DNA binding of the integrase, as well as several adjacent basic amino acids are solvent exposed. The identification of an SH3-like domain in integrase provides a new potential target for drug design.

Iterative procedure for structure determination from proton-proton NOEs using a full relaxation matrix approach. Application to a DNA octamer

Abstract A method is proposed, called the iterative relaxation matrix approach (IRMA), for the structure determination of biomolecules in solution based on 2D NOE data. Proton-proton distances are determined in a way in which indirect magnetization transfer (spin diffusion) is taken fully into account. In this method experimental NOEs are combined with calculated NOE values based on a molecular model. Back-transformation of this mixed NOE matrix gives a relaxation matrix which provides a better estimation of the cross-relaxation rates than can be obtained directly from the NOE cross peaks. From the cross-relaxation rates distance constraints can be derived, which are used in restrained molecular dynamics calculations to obtain an improved molecular model. The iteration cycle can be repeated until all experimental NOE values are satisfactorily explained. The method was tested with a DNA octamer, d(GCGTTGCG)·d(CGCAACGC).

Determination of biomolecular structures from proton-proton NOE's using a relaxation matrix approach

Abstract A method is proposed for the structure determination of biomolecules in solution based on 2D NOE data in which indirect magnetization transfer (spin diffusion) is taken into account. In this method experimental NOEs are supplemented with calculated NOEs based on a molecular model. Back transformation of this mixed NOE matrix gives a relaxation matrix which provides a better estimation for the cross-relaxation rates than can be obtained directly from the NOE cross-peaks. From the cross-relaxation rates distance constraints can be derived, which can be used in distance geometry and restrained molecular dynamics calculations to obtain an improved molecular model. The procedure can be repeated until all experimental NOEs are satisfactorily explained.

Structural and dynamic changes of photoactive yellow protein during its photocycle in solution.

Light irradiation of photoactive yellow protein (PYP) induces a photocycle, in which red-shifted (pR) and blue-shifted (pB) intermediates have been characterized. An NMR study of the long-lived pB intermediate now reveals that it exhibits a large degree of disorder and exists as a family of multiple conformers that exchange on a millisecond time scale. This shows that the behavior of PYP in solution is different from what has been observed in the crystalline state. Furthermore, differential refolding to ground state pG is observed, whereby the central β-sheet and parts of the helical structure are formed first and the region around the chromophore at a later stage.

The structure of the translational initiation factor IF1 from E.coli contains an oligomer‐binding motif

The structure of the translational initiation factor IF1 from Escherichia coli has been determined with multidimensional NMR spectroscopy. Using 1041 distance and 78 dihedral constraints, 40 distance geometry structures were calculated, which were refined by restrained molecular dynamics. From this set, 19 structures were selected, having low constraint energy and few constraint violations. The ensemble of 19 structures displays a root‐mean‐square deviation versus the average of 0.49 Å for the backbone atoms and 1.12 Å for all atoms for residues 6–36 and 46–67. The structure of IF1 is characterized by a five‐stranded β‐barrel. The loop connecting strands three and four contains a short 310 helix but this region shows considerably higher flexibility than the β‐barrel. The fold of IF1 is very similar to that found in the bacterial cold shock proteins CspA and CspB, the N‐terminal domain of aspartyl‐tRNA synthetase and the staphylococcal nuclease, and can be identified as the oligomer‐binding motif. Several proteins of this family are nucleic acid‐binding proteins. This suggests that IF1 plays its role in the initiation of protein synthesis by nucleic acid interactions. Specific changes of NMR signals of IF1 upon titration with 30S ribosomal subunit identifies several residues that are involved in the interaction with ribosomes.