John F. Hunt

The structural basis of cyclic diguanylate signal transduction by PilZ domains.

The second messenger cyclic diguanylate (c‐di‐GMP) controls the transition between motile and sessile growth in eubacteria, but little is known about the proteins that sense its concentration. Bioinformatics analyses suggested that PilZ domains bind c‐di‐GMP and allosterically modulate effector pathways. We have determined a 1.9 Å crystal structure of c‐di‐GMP bound to VCA0042/PlzD, a PilZ domain‐containing protein from Vibrio cholerae. Either this protein or another specific PilZ domain‐containing protein is required for V. cholerae to efficiently infect mice. VCA0042/PlzD comprises a C‐terminal PilZ domain plus an N‐terminal domain with a similar β‐barrel fold. C‐di‐GMP contacts seven of the nine strongly conserved residues in the PilZ domain, including three in a seven‐residue long N‐terminal loop that undergoes a conformational switch as it wraps around c‐di‐GMP. This switch brings the PilZ domain into close apposition with the N‐terminal domain, forming a new allosteric interaction surface that spans these domains and the c‐di‐GMP at their interface. The very small size of the N‐terminal conformational switch is likely to explain the facile evolutionary diversification of the PilZ domain.

Crystal structures of catalytic complexes of the oxidative DNA/RNA repair enzyme AlkB

Nucleic acid damage by environmental and endogenous alkylation reagents creates lesions that are both mutagenic and cytotoxic, with the latter effect accounting for their widespread use in clinical cancer chemotherapy. Escherichia coli AlkB and the homologous human proteins ABH2 and ABH3 (refs 5, 7) promiscuously repair DNA and RNA bases damaged by SN2 alkylation reagents, which attach hydrocarbons to endocyclic ring nitrogen atoms (N1 of adenine and guanine and N3 of thymine and cytosine). Although the role of AlkB in DNA repair has long been established based on phenotypic studies, its exact biochemical activity was only elucidated recently after sequence profile analysis revealed it to be a member of the Fe-oxoglutarate-dependent dioxygenase superfamily. These enzymes use an Fe(ii) cofactor and 2-oxoglutarate co-substrate to oxidize organic substrates. AlkB hydroxylates an alkylated nucleotide base to produce an unstable product that releases an aldehyde to regenerate the unmodified base. Here we have determined crystal structures of substrate and product complexes of E. coli AlkB at resolutions from 1.8 to 2.3u2009Å. Whereas the Fe-2-oxoglutarate dioxygenase core matches that in other superfamily members, a unique subdomain holds a methylated trinucleotide substrate into the active site through contacts to the polynucleotide backbone. Amide hydrogen exchange studies and crystallographic analyses suggest that this substrate-binding ‘lid’ is conformationally flexible, which may enable docking of diverse alkylated nucleotide substrates in optimal catalytic geometry. Different crystal structures show open and closed states of a tunnel putatively gating O2 diffusion into the active site. Exposing crystals of the anaerobic Michaelis complex to air yields slow but substantial oxidation of 2-oxoglutarate that is inefficiently coupled to nucleotide oxidation. These observations suggest that protein dynamics modulate redox chemistry and that a hypothesized migration of the reactive oxy-ferryl ligand on the catalytic Fe ion may be impeded when the protein is constrained in the crystal lattice.

Improving NMR Protein Structure Quality by Rosetta Refinement: A Molecular Replacement Study

The structure of human protein HSPC034 has been determined by both solution nuclear magnetic resonance (NMR) spectroscopy and X‐ray crystallography. Refinement of the NMR structure ensemble, using a Rosetta protocol in the absence of NMR restraints, resulted in significant improvements not only in structure quality, but also in molecular replacement (MR) performance with the raw X‐ray diffraction data using MOLREP and Phaser. This method has recently been shown to be generally applicable with improved MR performance demonstrated for eight NMR structures refined using Rosetta (Qian et al., Nature 2007;450:259–264). Additionally, NMR structures of HSPC034 calculated by standard methods that include NMR restraints have improvements in the RMSD to the crystal structure and MR performance in the order DYANA, CYANA, XPLOR‐NIH, and CNS with explicit water refinement (CNSw). Further Rosetta refinement of the CNSw structures, perhaps due to more thorough conformational sampling and/or a superior force field, was capable of finding alternative low energy protein conformations that were equally consistent with the NMR data according to the Recall, Precision, and F‐measure (RPF) scores. On further examination, the additional MR‐performance shortfall for NMR refined structures as compared with the X‐ray structure were attributed, in part, to crystal‐packing effects, real structural differences, and inferior hydrogen bonding in the NMR structures. A good correlation between a decrease in the number of buried unsatisfied hydrogen‐bond donors and improved MR performance demonstrates the importance of hydrogen‐bond terms in the force field for improving NMR structures. The superior hydrogen‐bond network in Rosetta‐refined structures demonstrates that correct identification of hydrogen bonds should be a critical goal of NMR structure refinement. Inclusion of nonbivalent hydrogen bonds identified from Rosetta structures as additional restraints in the structure calculation results in NMR structures with improved MR performance. Proteins 2009.

A general computational approach for repeat protein design

Repeat proteins have considerable potential for use as modular binding reagents or biomaterials in biomedical and nanotechnology applications. Here we describe a general computational method for building idealized repeats that integrates available family sequences and structural information with Rosetta de novo protein design calculations. Idealized designs from six different repeat families were generated and experimentally characterized; 80% of the proteins were expressed and soluble and more than 40% were folded and monomeric with high thermal stability. Crystal structures determined for members of three families are within 1Å root-mean-square deviation to the design models. The method provides a general approach for fast and reliable generation of stable modular repeat protein scaffolds.

S-Adenosylmethionine-dependent radical-based modification of biological macromolecules

Proteins and RNA molecules enjoy a variety of chemically complex post-translational and post-transcriptional modifications. The chemistry at work in these reactions, which was considered to be exclusively ionic in nature has recently been shown to depend on radical mechanisms in some cases. The overwhelming majority of these radical-based reactions are catalyzed by Radical-SAM enzymes. This review article highlights mechanistic and structural aspects of this class of reactions and indicates important research directions to be addressed.

Exploration of Alternate Catalytic Mechanisms and Optimization Strategies for Retroaldolase Design

Designed retroaldolases have utilized a nucleophilic lysine to promote carbon-carbon bond cleavage of β-hydroxy-ketones via a covalent Schiff base intermediate. Previous computational designs have incorporated a water molecule to facilitate formation and breakdown of the carbinolamine intermediate to give the Schiff base and to function as a general acid/base. Here we investigate an alternative active-site design in which the catalytic water molecule was replaced by the side chain of a glutamic acid. Five out of seven designs expressed solubly and exhibited catalytic efficiencies similar to previously designed retroaldolases for the conversion of 4-hydroxy-4-(6-methoxy-2-naphthyl)-2-butanone to 6-methoxy-2-naphthaldehyde and acetone. After one round of site-directed saturation mutagenesis, improved variants of the two best designs, RA114 and RA117, exhibited among the highest kcat (>10(-3)s(-1)) and kcat/KM (11-25M(-1)s(-1)) values observed for retroaldolase designs prior to comprehensive directed evolution. In both cases, the >10(5)-fold rate accelerations that were achieved are within 1-3 orders of magnitude of the rate enhancements reported for the best catalysts for related reactions, including catalytic antibodies (kcat/kuncat=10(6) to 10(8)) and an extensively evolved computational design (kcat/kuncat>10(7)). The catalytic sites, revealed by X-ray structures of optimized versions of the two active designs, are in close agreement with the design models except for the catalytic lysine in RA114. We further improved the variants by computational remodeling of the loops and yeast display selection for reactivity of the catalytic lysine with a diketone probe, obtaining an additional order of magnitude enhancement in activity with both approaches.

Target highlights in CASP9: Experimental target structures for the critical assessment of techniques for protein structure prediction.

One goal of the CASP community wide experiment on the critical assessment of techniques for protein structure prediction is to identify the current state of the art in protein structure prediction and modeling. A fundamental principle of CASP is blind prediction on a set of relevant protein targets, that is, the participating computational methods are tested on a common set of experimental target proteins, for which the experimental structures are not known at the time of modeling. Therefore, the CASP experiment would not have been possible without broad support of the experimental protein structural biology community. In this article, several experimental groups discuss the structures of the proteins which they provided as prediction targets for CASP9, highlighting structural and functional peculiarities of these structures: the long tail fiber protein gp37 from bacteriophage T4, the cyclic GMP‐dependent protein kinase Iβ dimerization/docking domain, the ectodomain of the JTB (jumping translocation breakpoint) transmembrane receptor, Autotaxin in complex with an inhibitor, the DNA‐binding J‐binding protein 1 domain essential for biosynthesis and maintenance of DNA base‐J (β‐D‐glucosyl‐hydroxymethyluracil) in Trypanosoma and Leishmania, an so far uncharacterized 73 residue domain from Ruminococcus gnavus with a fold typical for PDZ‐like domains, a domain from the phycobilisome core‐membrane linker phycobiliprotein ApcE from Synechocystis, the heat shock protein 90 activators PFC0360w and PFC0270w from Plasmodium falciparum, and 2‐oxo‐3‐deoxygalactonate kinase from Klebsiella pneumoniae. Proteins 2011;

Structural genomics reveals EVE as a new ASCH/PUA-related domain

We report on several proteins recently solved by structural genomics consortia, in particular by the Northeast Structural Genomics consortium (NESG). The proteins considered in this study differ substantially in their sequences but they share a similar structural core, characterized by a pseudobarrel five‐stranded beta sheet. This core corresponds to the PUA domain‐like architecture in the SCOP database. By connecting sequence information with structural knowledge, we characterize a new subgroup of these proteins that we propose to be distinctly different from previously described PUA domain‐like domains such as PUA proper or ASCH. We refer to these newly defined domains as EVE. Although EVE may have retained the ability of PUA domains to bind RNA, the available experimental and computational data suggests that both the details of its molecular function and its cellular function differ from those of other PUA domain‐like domains. This study of EVE and its relatives illustrates how the combination of structure and genomics creates new insights by connecting a cornucopia of structures that map to the same evolutionary potential. Primary sequence information alone would have not been sufficient to reveal these evolutionary links. Proteins 2009.

Structure and Dynamics of the P7 Protein from the Bacteriophage ϕ12

Cystoviruses are a class of enveloped double-stranded RNA viruses that use a multiprotein polymerase complex (PX) to replicate and transcribe the viral genome. Although the structures of the polymerase and ATPase components of the cystoviral PX are known and their functional behavior is understood to a large extent, no atomic-resolution structural information is available for the major capsid protein P1 that defines the overall structure and symmetry of the viral capsid and the essential protein P7. Toward obtaining a complete structural and functional understanding of the cystoviral PX, we have obtained the structure of P7 from the cystovirus phi 12 at a resolution of 1.8 A. The N-terminal core region (1-129) of P7 forms a novel homodimeric alpha/beta-fold having structural similarities with BRCT domains implicated in multiple protein-protein interactions in DNA repair proteins. Our results, combined with the known role of P7 in stabilizing the nucleation complex during capsid assembly, hint toward its participation in key protein-protein interactions within the cystoviral PX. Additionally, we have found through solution NMR studies that the C-terminal tail of P7 (130-169) that is essential for virus viability, although highly disordered, contains a nascent helix. We demonstrate for the first time, through NMR titrations, that P7 is capable of interacting with RNA. We find that both the N-terminal core and the dynamic C-terminal tail of P7 play a role in RNA recognition. This interaction leads to a significant reduction of the degree of disorder in the C-terminal tail. Given the requirement of P7 in maintaining genome packaging efficiency and transcriptional fidelity, our data suggest a central biological role for P7-RNA interactions.

The methylthiolation reaction mediated by the Radical-SAM enzymes

Over the past 10 years, considerable progress has been made in our understanding of the mechanistic enzymology of the Radical-SAM enzymes. It is now clear that these enzymes appear to be involved in a remarkably wide range of chemically challenging reactions. This review article highlights mechanistic and structural aspects of the methylthiotransferases (MTTases) sub-class of the Radical-SAM enzymes. The mechanism of methylthio insertion, now observed to be performed by three different enzymes is an exciting unsolved problem. This article is part of a Special Issue entitled: Radical SAM enzymes and Radical Enzymology.