Solution structure of Archaeglobus fulgidis peptidyl-tRNA hydrolase (Pth2) provides evidence for an extensive conserved family of Pth2 enzymes in archea, bacteria, and eukaryotes

The solution structure of protein AF2095 from the thermophilic archaea Archaeglobus fulgidis, a 123-residue (13.6-kDa) protein, has been determined by NMR methods. The structure of AF2095 is comprised of four alpha-helices and a mixed beta-sheet consisting of four parallel and anti-parallel beta-strands, where the alpha-helices sandwich the beta-sheet. Sequence and structural comparison of AF2095 with proteins from Homo sapiens, Methanocaldococcus jannaschii, and Sulfolobus solfataricus reveals that AF2095 is a peptidyl-tRNA hydrolase (Pth2). This structural comparison also identifies putative catalytic residues and a tRNA interaction region for AF2095. The structure of AF2095 is also similar to the structure of protein TA0108 from archaea Thermoplasma acidophilum, which is deposited in the Protein Data Bank but not functionally annotated. The NMR structure of AF2095 has been further leveraged to obtain good-quality structural models for 55 other proteins. Although earlier studies have proposed that the Pth2 protein family is restricted to archeal and eukaryotic organisms, the similarity of the AF2095 structure to human Pth2, the conservation of key active-site residues, and the good quality of the resulting homology models demonstrate a large family of homologous Pth2 proteins that are conserved in eukaryotic, archaeal, and bacterial organisms, providing novel insights in the evolution of the Pth and Pth2 enzyme families.     

Clustering algorithms for identifying core atom sets and for assessing the precision of protein structure ensembles

An important open question in the field of NMR-based biomolecular structure determination is how best to characterize the precision of the resulting ensemble of structures. Typically, the RMSD, as minimized in superimposing the ensemble of structures, is the preferred measure of precision. However, the presence of poorly determined atomic coordinates and multiple "RMSD-stable domains"--locally well-defined regions that are not aligned in global superimpositions--complicate RMSD calculations. In this paper, we present a method, based on a novel, structurally defined order parameter, for identifying a set of core atoms to use in determining superimpositions for RMSD calculations. In addition we present a method for deciding whether to partition that core atom set into "RMSD-stable domains" and, if so, how to determine partitioning of the core atom set. We demonstrate our algorithm and its application in calculating statistically sound RMSD values by applying it to a set of NMR-derived structural ensembles, superimposing each RMSD-stable domain (or the entire core atom set, where appropriate) found in each protein structure under consideration. A parameter calculated by our algorithm using a novel, kurtosis-based criterion, the epsilon-value, is a measure of precision of the superimposition that complements the RMSD. In addition, we compare our algorithm with previously described algorithms for determining core atom sets. The methods presented in this paper for biomolecular structure superimposition are quite general, and have application in many areas of structural bioinformatics and structural biology.     

Solution NMR structure of CD1104B from pathogenic Clostridium difficile reveals a distinct α-helical architecture and provides first structural representative of protein domain family PF14203

A high-quality structure of the 68-residue protein CD1104B from Clostridium difficile strain 630 exhibits a distinct all α-helical fold. The structure presented here is the first representative of bacterial protein domain family PF14203 (currently 180 members) of unknown function (DUF4319) and reveals that the side-chains of the only two strictly conserved residues (Glu 8 and Lys 48) form a salt bridge. Moreover, these two residues are located in the vicinity of the largest surface cleft which is predicted to contribute to a surface area involved in protein–protein interactions. This, along with its coding in transposon CTn4, suggests that CD1104B (and very likely all members of Pfam 14203) functions by interacting with other proteins required for the transfer of transposons between different bacterial species.     

Solution NMR and X-ray crystal structures of membrane-associated Lipoprotein-17 domain reveal a novel fold

The conserved Lipoprotein-17 domain of membrane-associated protein Q9PRA0_UREPA from Ureaplasma parvum was selected for structure determination by the Northeast Structural Genomics Consortium, as part of the Protein Structure Initiative's program on structure-function analysis of protein domains from large domain sequence families lacking structural representatives. The 100-residue Lipoprotein-17 domain is a "domain of unknown function" (DUF) that is a member of Pfam protein family PF04200, a large domain family for which no members have characterized biochemical functions. The three-dimensional structure of the Lipoprotein-17 domain of protein Q9PRA0_UREPA was determined by both solution NMR and by X-ray crystallography at 2.5 ?. The two structures are in good agreement with each other. The domain structure features three α-helices, α1 through α3, and five β-strands. Strands β1/β2, β3/β4, β4/β5 are anti-parallel to each other. Strands β1and β2 are orthogonal to strands β3, β4, β5, while helix α3 is formed between the strands β3 and β4. One-turn helix α2 is formed between the strands β1 and β2, while helix α1 occurs in the N-terminal polypeptide segment. Searches of the Protein Data Bank do not identify any other protein with significant structural similarity to Lipoprotein-17 domain of Q9PRA0_UREPA, indicating that it is a novel protein fold.     

The Use of the Condensed Single Protein Production System for Isotope-Labeled Outer Membrane Proteins,OmpA and OmpX in <Emphasis Type="Italic">E. coli</Emphasis>

Gram-negative bacteria consist of two independent membranes, the inner cytoplasmic membrane and the outer membrane. The outer membrane contains a number of β-barrel proteins such as OmpF, OmpC, OmpA, and OmpX. In this article, we explored to use the condensed Single Protein Production (cSPP) system for isotope labelling of OmpA and OmpX for NMR structural study, both of which are known to consist of eight β-strands forming a barrel in the outer membrane. Using a deletion strain lacking all major outer membrane proteins, both OmpA and OmpX were expressed well in a 20-fold cSPP system. We demonstrated that outer membrane fractions prepared from the cSPP system in M9 medium containing ¹⁵N–NH₄Cl can be directly used for NMR structural study of the outer mebrane proteins without any further purification to get excellent [¹H–¹⁵N]-TROSY spectra. This method would be quite valuable for the study of pure proteins in their native membrane environment without the need of purification and reconstitution.     

Solution structure of 4'-phosphopantetheine - GmACP3 from Geobacter metallireducens: a specialized acyl carrier protein with atypical structural features and a putative role in lipopolysaccharide biosynthesis

GmACP3 from Geobacter metallireducens is a specialized acyl carrier protein (ACP) whose gene, gmet_2339, is located near genes encoding many proteins involved in lipopolysaccharide (LPS) biosynthesis, indicating a likely function for GmACP3 in LPS production. By overexpression in Escherichia coli, about 50% holo-GmACP3 and 50% apo-GmACP3 were obtained. Apo-GmACP3 exhibited slow precipitation and non-monomeric behavior by (15)N NMR relaxation measurements. Addition of 4'-phosphopantetheine (4'-PP) via enzymatic conversion by E. coli holo-ACP synthase resulted in stable >95% holo-GmACP3 that was characterized as monomeric by (15)N relaxation measurements and had no indication of conformational exchange. We have determined a high-resolution solution structure of holo-GmACP3 by standard NMR methods, including refinement with two sets of NH residual dipolar couplings, allowing for a detailed structural analysis of the interactions between 4'-PP and GmACP3. Whereas the overall four helix bundle topology is similar to previously solved ACP structures, this structure has unique characteristics, including an ordered 4'-PP conformation that places the thiol at the entrance to a central hydrophobic cavity near a conserved hydrogen-bonded Trp-His pair. These residues are part of a conserved WDSLxH/N motif found in GmACP3 and its orthologs. The helix locations and the large hydrophobic cavity are more similar to medium- and long-chain acyl-ACPs than to other apo- and holo-ACP structures. Taken together, structural characterization along with bioinformatic analysis of nearby genes suggests that GmACP3 is involved in lipid A acylation, possibly by atypical long-chain hydroxy fatty acids, and potentially is involved in synthesis of secondary metabolites.     

Suppression of phospholipid biosynthesis by cerulenin in the condensed Single-Protein-Production (cSPP) system

Using the single-protein-production (SPP) system, a protein of interest can be exclusively produced in high yield from its ACA-less gene in Escherichia coli expressing MazF, an ACA-specific mRNA interferase. It is thus feasible to study a membrane protein by solid-state NMR (SSNMR) directly in natural membrane fractions. In developing isotope-enrichment methods, we observed that ¹³C was also incorporated into phospholipids, generating spurious signals in SSNMR spectra. Notable, with the SPP system a protein can be produced in total absence of cell growth caused by antibiotics. Here, we demonstrate that cerulenin, an inhibitor of phospholipid biosynthesis, can suppress isotope incorporation in the lipids without affecting membrane protein yield in the SPP system. SSNMR analysis of ATP synthase subunit c, an E. coli inner membrane protein, produced by the SPP method using cerulenin revealed that ¹³C resonance signals from phospholipid were markedly reduced, while signals for the isotope-enriched protein were clearly present.