Refined crystal structure of ferredoxin II from Desulfovibrio gigas at 1.7 A.

The crystal structure of ferredoxin II from Desulfovibrio gigas has been determined using phasing from anomalous scattering data at a resolution of 1.7 A and refined to an R-factor of 0.157. The molecule has an overall chain fold similar to that of the other bacterial ferredoxins of known structure. The molecule contains a single 3Fe-4S cluster with geometry indistinguishable from the 4Fe-4S clusters, and a disulfide bond near the site corresponding to the position of the second cluster of two-cluster ferredoxins. The cluster is bound by cysteine residues 8, 14 and 50. The side-chain of cysteine 11 extends away from the cluster, but could rotate to become the fourth cysteine ligand in the four-iron form of the molecule given a local adjustment of the polypeptide chain. This residue is modified, however, by what appears to be a methanethiol group. There are a total of eight NH . . . S bonds to the inorganic and cysteine sulfur atoms of the Fe-S cluster. There is an additional residue found that is not reported for the chemical sequence: according to the electron density a valine residue should be inserted after residue 55.

Protein serine/threonine phosphatases.

In the past year, the three-dimensional structures of two serine/threonine phosphatases, protein phosphatase-1 and protein phosphatase-2b (calcineurin), have been determined. The new information puts previous sequence comparisons and mutagenesis studies into a detailed structural perspective. The active-site structure and catalytic mechanism appear to be common to a variety of phosphoesterase enzymes.

Deposit3D: a tool for automating structure depositions to the Protein Data Bank.

Almost all successful protein structure-determination projects in the public sector culminate in a structure deposition to the Protein Data Bank (PDB). In order to expedite the deposition process, Deposit3D has been developed. This command-line script calculates or gathers all the required structure-deposition information and outputs this data into a mmCIF file for subsequent upload through the RCSB PDB ADIT interface. Deposit3D might be particularly useful for structural genomics pipeline projects because it allows workers involved with various stages of a structure-determination project to pool their different categories of annotation information before starting a deposition session.

Structural studies of the engrailed homeodomain.

The structure of the Drosophila engrailed homeodomain has been solved by molecular replacement and refined to an R-factor of 19.7% at a resolution of 2.1 A. This structure offers a high-resolution view of an important family of DNA-binding proteins and allows comparison to the structure of the same protein bound to DNA. The most significant difference between the current structure and that of the 2.8-A engrailed-DNA complex is the close packing of an extended strand against the rest of the protein in the unbound protein. Structural features of the protein not previously noted include a "herringbone" packing of 4 aromatic residues in the core of the protein and an extensive network of salt bridges that covers much of the helix 1-helix 2 surface. Other features that may play a role in stabilizing the native state include the interaction of buried carbonyl oxygen atoms with the edge of Phe 49 and a bias toward statistically preferred side-chain dihedral angles. There is substantial disorder at both ends of the 61 amino acid protein. A 51-amino acid variant of engrailed (residues 6-56) was synthesized and shown by CD and thermal denaturation studies to be structurally and thermodynamically similar to the full-length domain.

The crystal structure of the three-iron ferredoxin II from Desulfovibrio gigas.

The crystal structure of oxidized ferredoxin II from the sulfate-reducing bacterium Desulfovibrio gigas has been determined and refined at 1.7 A resolution. The folding of the polypeptide chain is similar to that of the 2[4Fe-4S] ferredoxin in Peptococcus aerogenes, except for an extended helical segment near the C-terminus. The single [3Fe-4S] cluster in D. gigas is similar to a [4Fe-4S] cluster, but lacks one Fe atom and is coordinated to Cys-8, -14 and -50. The side chain of Cys-11 is not bound to the cluster, but is rotated toward the solvent and modified by some, as yet undetermined, chemical group. Cys-18 and Cys-42 form a disulfide bridge. A previously undetected extra amino acid is found after residue 55.

Structure-based design of novel, urea-containing FKBP12 inhibitors.

The structure-based design and subsequent chemical synthesis of novel, urea-containing FKBP12 inhibitors are described. These compounds are shown to disrupt the cis-trans peptidylprolyl isomerase activity of FKBP12 with inhibition constants (Ki,app) approaching 0.10 microM. Analyses of several X-ray crystal structures of FKBP12-urea complexes demonstrate that the urea-containing inhibitors associate with FKBP12 in a manner that is similar to, but significantly different from, that observed for the natural product FK506.

Intrinsically disordered protein.

Proteins can exist in a trinity of structures: the ordered state, the molten globule, and the random coil. The five following examples suggest that native protein structure can correspond to any of the three states (not just the ordered state) and that protein function can arise from any of the three states and their transitions. (1) In a process that likely mimics infection, fd phage converts from the ordered into the disordered molten globular state. (2) Nucleosome hyperacetylation is crucial to DNA replication and transcription; this chemical modification greatly increases the net negative charge of the nucleosome core particle. We propose that the increased charge imbalance promotes its conversion to a much less rigid form. (3) Clusterin contains an ordered domain and also a native molten globular region. The molten globular domain likely functions as a proteinaceous detergent for cell remodeling and removal of apoptotic debris. (4) In a critical signaling event, a helix in calcineurin becomes bound and surrounded by calmodulin, thereby turning on calcineurin's serine/threonine phosphatase activity. Locating the calcineurin helix within a region of disorder is essential for enabling calmodulin to surround its target upon binding. (5) Calsequestrin regulates calcium levels in the sarcoplasmic reticulum by binding approximately 50 ions/molecule. Disordered polyanion tails at the carboxy terminus bind many of these calcium ions, perhaps without adopting a unique structure. In addition to these examples, we will discuss 16 more proteins with native disorder. These disordered regions include molecular recognition domains, protein folding inhibitors, flexible linkers, entropic springs, entropic clocks, and entropic bristles. Motivated by such examples of intrinsic disorder, we are studying the relationships between amino acid sequence and order/disorder, and from this information we are predicting intrinsic order/disorder from amino acid sequence. The sequence-structure relationships indicate that disorder is an encoded property, and the predictions strongly suggest that proteins in nature are much richer in intrinsic disorder than are those in the Protein Data Bank. Recent predictions on 29 genomes indicate that proteins from eucaryotes apparently have more intrinsic disorder than those from either bacteria or archaea, with typically > 30% of eucaryotic proteins having disordered regions of length > or = 50 consecutive residues.

Structure of WF-3681, 3-(2,5-dihydro-4-hydroxy-5-oxo-3-phenyl-2-furyl)propionic acid.

C13H12O5, Mr = 248.24, monoclinic, C2/c, a = 18.757 (8), b = 7.282 (2), c = 17.511 (8) A, beta = 91.20 (3) degrees, V = 2391 (3) A3, Z = 8, Dx = 1.379 Mg m-3, lambda(Cu K alpha) = 1.54178 A, mu = 0.859 mm-1, F(000) = 1040, T = 293 K. Final R = 0.054 for 1409 unique observed reflections. The molecule contains two planar regions which differ in orientation by 5.7 degrees. Distances from the carbonyl carbons to the center of the phenyl ring are not in the range found in the crystal structures of other potent aldose reductase inhibitor molecules.

Rapid automated molecular replacement by evolutionary search.

A new procedure for molecular replacement is presented in which an efficient six-dimensional search is carried out using an evolutionary optimization algorithm. In this procedure, a population of initially random molecular-replacement solutions is iteratively optimized with respect to the correlation coefficient between observed and calculated structure factors. The sensitivity and reliability of the method is enhanced by uniform sampling of the rotational-search space and the use of continuously variable rotational and translational parameters. The process is several orders of magnitude faster than a systematic six-dimensional search, and comparisons show that it can identify solutions using significantly less accurate or less complete search models than is possible with two existing molecular-replacement methods. A program incorporating the method, EPMR, allows the rapid and highly automated solution of molecular-replacement problems involving single or multiple molecules in the asymmetric unit. EPMR has been used to solve a number of difficult molecular-replacement problems.

Crystallization and preliminary X-ray diffraction studies of the engrailed homeodomain and of an engrailed homeodomain/DNA complex.

The homeodomain from the engrailed protein of Drosophila has been crystallized from ammonium phosphate at pH 6.8. The crystals form in space group P6(1)22 (or P6(5)22), with cell dimensions a = b = 44.8 A and c = 118.2 A. These crystals diffract to 1.8 A resolution. A complex containing the engrailed homeodomain and a duplex DNA site also has been crystallized. The cocrystals form in space group C2 with a = 131.2 A, b = 45.5 A, c = 72.9 A and beta = 119.0 degrees. These crystals diffract to 2.6 A resolution.

Molecular replacement by evolutionary search.

Stochastic search algorithms can be used to perform rapid six-dimensional molecular-replacement searches. A molecular-replacement procedure has been developed that uses an evolutionary algorithm to simultaneously optimize the orientation and position of a search model in a unit cell. Here, the performance of this algorithm and its dependence on search model quality and choice of target function are examined. Although the evolutionary search procedure is capable of finding solutions with search models that represent only a small fraction of the total scattering matter of the target molecule, the efficiency of the search procedure is highly dependent on the quality of the search model. Polyalanine models frequently provide better search efficiency than all-atom models, even in cases where the side-chain positions are known with high accuracy. Although the success of the search procedure is not highly dependent on the statistic used as the target function, the correlation coefficient between observed and calculated structure-factor amplitudes generally results in better search efficiency than does the R factor. An alternative stochastic search procedure, simulated annealing, provides similar overall performance to evolutionary search. Methods of extending the evolutionary search algorithm to include internal optimization, selection and construction of the search model are now beginning to be investigated.

Thousands of proteins likely to have long disordered regions.

Neural network predictors of protein disorder using primary sequence information were developed and applied to the Swiss Protein Database. More than 15,000 proteins were predicted to contain disordered regions of at least 40 consecutive amino acids, with more than 1,000 having especially high scores indicating disorder. These results support proposals that consideration of structure-activity relationships in proteins need to be broadened to include unfolded or disordered protein.

Crystal structure of an engrailed homeodomain-DNA complex at 2.8 A resolution: a framework for understanding homeodomain-DNA interactions.

The crystal structure of a complex containing the engrailed homeodomain and a duplex DNA site has been determined at 2.8 A resolution and refined to a crystallographic R factor of 24.4%. In this complex, two separate regions of the 61 amino acid polypeptide contact a TAAT subsite. An N-terminal arm fits into the minor groove, and the side chains of Arg-3 and Arg-5 make contacts near the 5' end of this "core consensus" binding site. An alpha helix fits into the major groove, and the side chains of IIe-47 and Asn-51 contact base pairs near the 3' end of the TAAT site. This "recognition helix" is part of a structurally conserved helix-turn-helix unit, but these helices are longer than the corresponding helices in the lambda repressor, and the relationship between the helix-turn-helix unit and the DNA is significantly different.

Crystal structures of human calcineurin and the human FKBP12-FK506-calcineurin complex.

Calcineurin (CaN) is a calcium- and calmodulin-dependent protein serine/threonine phosphate which is critical for several important cellular processes, including T-cell activation. CaN is the target of the immunosuppressive drugs cyclosporin A and FK506, which inhibit CaN after forming complexes with cytoplasmic binding proteins (cyclophilin and FKBP12, respectively). We report here the crystal structures of full-length human CaN at 2.1 A resolution and of the complex of human CaN with FKBP12-FK506 at 3.5 A resolution. In the native CaN structure, an auto-inhibitory element binds at the Zn/Fe-containing active site. The metal-site geometry and active-site water structure suggest a catalytic mechanism involving nucleophilic attack on the substrate phosphate by a metal-activated water molecule. In the FKBP12-FK506-CaN complex, the auto-inhibitory element is displaced from the active site. The site of binding of FKBP12-FK506 appears to be shared by other non-competitive inhibitors of calcineurin, including a natural anchoring protein.

Interaction of a novel GDP exchange inhibitor with the Ras protein.

Mutated, tumorigenic Ras is present in a variety of human tumors. Compounds that inhibit tumorigenic Ras function may be useful in the treatment of Ras-related tumors. The interaction of a novel GDP exchange inhibitor (SCH-54292) with the Ras-GDP protein was studied by NMR spectroscopy. The binding of the inhibitor to the Ras protein was enhanced at low Mg2+ concentrations, which enabled the preparation of a stable complex for NMR study. To understand the enhanced inhibitor binding and the increased GDP dissociation rates of the Ras protein, the conformational changes of the Ras protein at low Mg2+ concentrations was investigated using two-dimensional 1H-15N HSQC experiments. The Ras protein existed in two conformations in slow exchange on the NMR time scale under such conditions. The conformational changes mainly occurred in the GDP binding pocket, in the switch I and the switch II regions, and were reversible. The Ras protein resumed its regular conformation after an excess amount of Mg2+ was added. A model of the inhibitor in complex with the Ras-GDP protein was derived from intra- and intermolecular NOE distance constraints, and revealed that the inhibitor bound to the critical switch II region of the Ras protein.