research advances
April 2009 research highlight
The center cut
PSI-SGKB [doi:10.1038/rhighlts_psisgkb.2009.21]
Directors of the PSI centers highlight their choice of important structural genomics studies.
Ian Wilson
Director, PSI JCSG
The SARS unique domain
The 1922-residue non-structural protein 3 (nsp3) of the severe acute respiratory syndrome coronavirus (SARS-CoV) contains a continuous stretch of 400 amino-acid residues with no apparent relationship to any other protein; in contrast, the remainder of the nsp3 sequence does, to a certain degree, have similarity to proteins in other coronaviruses. Biochemical dissection of this 'SARS-unique domain' (SUD) and the NMR structure of a globular domain contained therein shows that the SUD contains a macrodomain fold with similarity to poly-ADP-ribose-binding proteins, histone-associated proteins and helicases. Intriguingly, the closest structural homolog is a neighboring domain of nsp3, characterized as an ADP-ribose-1''-phosphatase. NMR experiments and biochemical assays uncovered a cavity on the protein surface that binds poly(A) and other RNA sequences, suggesting a function related to viral replication, possibly regulation of replication initiation by binding to poly(A) tails. The presence of this newly characterized macrodomain is a key difference between SARS-CoV and other coronaviruses characterized so far, and it is tempting to speculate that the more severe symptoms of SARS may be related to the presence of the SUD.
Y-d-glutamyl-l-diamino acid endopeptidase
This study describes the structure determination and analysis of an enzyme containing a bacterial SH3 domain and a ubiquitous cysteine peptidase CHAP domain. This is the first structural representative of a large class of bacterial cell-wall peptidases. Moreover, the authors were able to correctly infer the enzymatic activity from the structure. New insights were obtained regarding the substrate specificity that is defined by these conserved structural modules.
A novel Sm-like protein
This paper presents a first detailed analysis of a protein representing novel, previously unknown protein families found in the marine metagenomics projects of Craig Venter. The PSI JCSG solved the structure of the ECX21941 protein, a representative of a very large family of metagenomics sequences, having no detectable similarity to any previously characterized protein family. Structural analysis of the ECX21941 protein shows that this family represents a diverging branch of the Sm/LSm RNA-binding protein family. The structure also illustrates a new pentameric assembly of Sm/LSm monomers and novel structural features that suggest that the specific, and as yet unknown, function of the ECX21941 protein and homologs may be different from currently characterized members of the Sm/LSm family. This interplay between structural conservation and sequence and functional divergence is a characteristic of most metagenomics proteins studied so far.
Guy Montelione
Director, PSI NESG
Alg13: a sugar donor subunit
The study presents the solution structure of Alg13, the glycosyl donor-binding domain of an important bipartite glycosyltransferase in the yeast Saccharomyces cerevisiae. This glycosyltransferase is unusual in that it is active only in the presence of a binding partner, Alg14. Alg13 is found to adopt a unique topology among glycosyltransferases. Rather than the conventional Rossmann fold found in all GT-B enzymes, the N-terminal half of the protein is a Rossmann-like fold with a mixed parallel and antiparallel β-sheet.The Rossmann fold of the C-terminal half of Alg13 is conserved. However, although conventional GT-B enzymes usually possess three helices at the C terminus, only two helices are present in Alg13. Titration of Alg13 with both UDP-GlcNAc, the native glycosyl donor, and a paramagnetic mimic, UDP-TEMPO, shows that the interaction of Alg13 with the sugar donor is primarily through the residues in the C-terminal half of the protein.
NMR breakthrough
A significant breakthrough in NMR data collection is described, providing information on how to obtain clean, high-resolution NMR spectra without the need for phase correction. This is particularly valuable for studies of larger proteins and membrane proteins with high chemical shift overlap and for the development of automated methods of NMR data analysis.
The contribution of structural genomics
The value of PSI protein structures in providing templates for homology modeling is described in this important paper. The second phase of PSI (2005 to now) contributed some 8% of all structures deposited in the PDB and more than 20% of all novel structures (that is, structures for protein sequences with no structural representative in the PDB on the date of deposition). Metrics are defined for estimating the modeling leverage and coverage of a newly deposited protein structure. According to these metrics, the PSI has provided more than 300,000 protein sequences that could not be modeled with templates available in the PDB on the dates the PSI structures were deposited. The analysis shows that some 70% of novel leverage provided by structures determined by US structural biology groups in recent years came from the NIH PSI groups.
Modeling workshop
Although different applications require different accuracies of homology modeling, the wide range of accuracy provided by current modeling methods provides structures that can have a high impact on the design of experiments and for understanding biology. This summary of a workshop to discuss the value of homology models addresses the question “what good are homology models”. The answer is it depends on what you want them for, but for many applications they are very valuable.
Stephen Burley
Director, PSI NYSGXRC
Mysterious archaea
Uncovering the evolutionary relationships among the three domains of life (archaea, eubacteria and eukaryotes) constitutes one of the great challenges for post-genomic biology and they are still not well understood. Of the three domains, archaea remain the biggest mystery. They are also of interest as they resemble both eubacteria and eukaryotes in some respects and hold considerable promise for the biotechnology industry. Many archaeal organisms are thermophilic and some even survive at temperatures > >100°C, and represent the only known strictly anerobic methanogens on the planet. In this study, we analyze the crystal structures of four members of the archaeal UPF0201 protein family. Unexpectedly, these structures proved to be similar to those of the ribosomal L5 proteins, which are responsible for binding to 5S RNA. In addition to comparing and contrasting the four UPF0201 protein structures, we have used structure-based sequence alignments to construct a phylogenetic tree that relates UPF0201 family members to L5 ribosomal subunits and other structurally similar RNA binding proteins, thereby extending the evolutionary purview of the RRM motif superfamily.
Andrzej Joachimiak
Director, PSI MCSG
Fructose-1,6-bisphosphatase
Gluconeogenesis is an important metabolic pathway that produces glucose from noncarbohydrate precursors such as organic acids, fatty acids, amino acids or glycerol. Fructose-1,6-bisphosphatase, a key enzyme of gluconeogenesis, is found in all organisms, and five different classes of these enzymes have been identified. In this study we demonstrate that Escherichia coli has two class II fructose-1,6-bisphosphatases, GlpX and YggF, which show different catalytic properties. We present the first crystal structure of a class II fructose-1,6-bisphosphatase (GlpX) determined in a free state and in the complex with a substrate (fructose 1,6-bisphosphate) or inhibitor (phosphate). The crystal structure of the ligand-free GlpX revealed a compact, globular shape with two α/β-sandwich domains. The core fold of GlpX is structurally similar to that of Li+-sensitive phosphatases implying that they have a common evolutionary origin and catalytic mechanism. The structure of the GlpX complex with fructose 1,6-bisphosphate revealed that the active site is located between two domains and accommodates several conserved residues coordinating two metal ions and the substrate. The third metal ion is bound to phosphate 6 of the substrate. Inorganic phosphate strongly inhibited activity of both GlpX and YggF, and the crystal structure of the GlpX complex with phosphate demonstrated that the inhibitor molecule binds to the active site. Alanine-replacement mutagenesis of GlpX identified 12 conserved residues important for activity and suggested that Thr90 is the primary catalytic residue. Our data provide insight into the molecular mechanisms of the substrate specificity and catalysis of GlpX and other class II fructose-1,6-bisphosphatases.
System 48
In this study we describe a plate-based cloning and expression strategy for efficient high-throughput generation of validated expression clones in Escherichia coli. The process incorporates 48- or 96-well plates at all stages, including the cloning and colony selection phases, that are often performed manually. A 48-grid agar growth plate has been integrated into the colony selection component to improve throughput at the cloning stage. The combinations of 48- and 96-well plate formats are compatible with automated liquid handlers and multichannel pipettes. This revised cloning and expression pipeline increases throughput significantly, and also results in a reduction in both time and material requirements. The system has been validated by the production and screening of several thousand clones at the PSI MSCG.
Phospholipid synthesis
PlsX is a key enzyme that coordinates the production of fatty acids and membrane phospholipids. The plsX gene is co-localized with a bacterial fab gene cluster that encodes several key fatty-acid biosynthetic enzymes. The protein is a member of a large, conserved protein family (Pfam02504) found exclusively in bacteria. The PlsX sequence homologs include both phosphate acetyltransferases and phosphate butaryltransferases that catalyze the transfer of an acetyl or butaryl group to orthophosphate. We have determined the crystal structure of PlsX from the human pathogen Enterococcus faecalis. PlsX is an α/β/α sandwich that resembles a Rossmann fold and forms a dimer. A putative catalytic site has been identified within a deep groove on the interface between monomers. This site showed strong surface similarity to epimerases and reductases. It was recently proposed that PlsX is a phosphate acyltransferase that catalyzes the formation of acyl-phosphate from the acyl-acyl carrier protein; however, the specific biochemical function of the PlsX protein awaits further experimental scrutiny.
