Anthony Semesi

An NMR approach to structural proteomics

The influx of genomic sequence information has led to the concept of structural proteomics, the determination of protein structures on a genome-wide scale. Here we describe an approach to structural proteomics of small proteins using NMR spectroscopy. Over 500 small proteins from several organisms were cloned, expressed, purified, and evaluated by NMR. Although there was variability among proteomes, overall 20% of these proteins were found to be readily amenable to NMR structure determination. NMR sample preparation was centralized in one facility, and a distributive approach was used for NMR data collection and analysis. Twelve structures are reported here as part of this approach, which allowed us to infer putative functions for several conserved hypothetical proteins.

Strategies for structural proteomics of prokaryotes: Quantifying the advantages of studying orthologous proteins and of using both NMR and X-ray crystallography approaches

Only about half of non‐membrane‐bound proteins encoded by either bacterial or archaeal genomes are soluble when expressed in Escherichia coli (Yee et al., Proc Natl Acad Sci USA 2002;99:1825–1830 ; Christendat et al., Prog Biophys Mol Biol 200;73:339–345) . This property limits genome‐scale functional and structural proteomics studies, which depend on having a recombinant, soluble version of each protein. An emerging strategy to increase the probability of deriving a soluble derivative of a protein is to study different sequence homologues of the same protein, including representatives from thermophilic organisms, based on the assumption that the stability of these proteins will facilitate structural analysis. To estimate the relative merits of this strategy, we compared the recombinant expression, solubility, and suitability for structural analysis by NMR and/or X‐ray crystallography for 68 pairs of homologous proteins from E. coli and Thermotoga maritima. A sample suitable for structural studies was obtained for 62 of the 68 pairs of homologs under standardized growth and purification procedures. Fourteen (eight E. coli and six T. maritima proteins) samples generated NMR spectra of a quality suitable for structure determination and 30 (14 E. coli and 16 T. maritima proteins) samples formed crystals. Only three (one E. coli and two T. maritima proteins) samples both crystallized and had excellent NMR properties. The conclusions from this work are: (1) The inclusion of even a single ortholog of a target protein increases the number of samples for structural studies almost twofold; (2) there was no clear advantage to the use of thermophilic proteins to generate samples for structural studies; and (3) for the small proteins analyzed here, the use of both NMR and crystallography approaches almost doubled the number of samples for structural studies. Proteins 2003;50:392–399.

NMR structure and binding studies confirm that PA4608 from Pseudomonas aeruginosa is a PilZ domain and a c‐di‐GMP binding protein

PA4608 is a 125 residue protein from Pseudomonas aeruginosa with a recent identification as a PilZ domain and putative bis‐(3′‐5′)‐cyclic dimeric guanosine monophosphate (c‐di‐GMP) adaptor protein that plays a role in bacterial second‐messenger regulated processes. The nuclear magnetic resonance (NMR) structure of PA4608 has been determined and c‐di‐GMP binding has been confirmed by NMR titration studies. The monomeric core structure of PA4608 contains a six‐stranded anti‐parallel β barrel flanked by three helices. Conserved surface residues among PA4608 homologs suggest the c‐di‐GMP binding site is at one end of the barrel and includes residues in the helices as well as in the unstructured N‐terminus. Chemical shift changes in PA4608 resonances upon titration with c‐di‐GMP confirm binding. This evidence supports the hypothesis that proteins containing PilZ domains are the long‐sought c‐di‐GMP adaptor proteins. Proteins 2007.

NleG Type 3 Effectors from Enterohaemorrhagic Escherichia coli Are U-Box E3 Ubiquitin Ligases

NleG homologues constitute the largest family of type 3 effectors delivered by pathogenic E. coli, with fourteen members in the enterohaemorrhagic (EHEC) O157:H7 strain alone. Identified recently as part of the non-LEE-encoded (Nle) effector set, this family remained uncharacterised and shared no sequence homology to other proteins including those of known function. The C-terminal domain of NleG2-3 (residues 90 to 191) is the most conserved region in NleG proteins and was solved by NMR. Structural analysis of this structure revealed the presence of a RING finger/U-box motif. Functional assays demonstrated that NleG2-3 as well as NleG5-1, NleG6-2 and NleG9′ family members exhibited a strong autoubiquitination activity in vitro; a characteristic usually expressed by eukaryotic ubiquitin E3 ligases. When screened for activity against a panel of 30 human E2 enzymes, the NleG2-3 and NleG5-1 homologues showed an identical profile with only UBE2E2, UBE2E3 and UBE2D2 enzymes supporting NleG activity. Fluorescence polarization analysis yielded a binding affinity constant of 56±2 µM for the UBE2D2/NleG5-1 interaction, a value comparable with previous studies on E2/E3 affinities. The UBE2D2 interaction interface on NleG2-3 defined by NMR chemical shift perturbation and mutagenesis was shown to be generally similar to that characterised for human RING finger ubiquitin ligases. The alanine substitutions of UBE2D2 residues Arg5 and Lys63, critical for activation of eukaryotic E3 ligases, also significantly decreased both NleG binding and autoubiquitination activity. These results demonstrate that bacteria-encoded NleG effectors are E3 ubiquitin ligases analogous to RING finger and U-box enzymes in eukaryotes.

Solution structure of the yeast ubiquitin-like modifier protein Hub1

AbstractabbreviationsUBL, ubiquitin-like modifier; Saccharomyces cerevisiae, S. cerevisiae; Eschericia coli, E. coli; NMR, nuclear magnetic resonance; NOE, nuclear Overhauser enhancement; NOESY, NOE spectroscopy; TOCSY, total correlated spectroscopy.

The crystal structure of hypothetical protein MTH1491 from Methanobacterium thermoautotrophicum.

As part of our structural proteomics initiative, we have determined the crystal structure of MTH1491, a previously uncharacterized hypothetical protein from Methanobacterium thermoautotrophicum. MTH1491 is one of numerous structural genomics targets selected in a genome‐wide survey of uncharacterized proteins. It belongs to a family of proteins whose biological function is not known. The crystal structure of MTH1491, the first structure for this family of proteins, consists of an overall five‐stranded parallel β‐sheet with strand order 51234 and flanking helices. The oligomeric form of this molecule is a trimer as seen from both crystal contacts and gel filtration studies. Analysis revealed that the structure of MTH1491 is similar to that of dehydrogenases, amidohydrolases, and oxidoreductases. Using a combination of sequence and structural analyses, we showed that MTH1491 does not belong to either the dehydrogenase or the amidohydrolase superfamilies of proteins.

Solution structure of ribosomal protein S28E from Methanobacterium thermoautotrophicum

The ribosomal protein S28E from the archaeon Methanobacterium thermoautotrophicum is a component of the 30S ribosomal subunit. Sequence homologs of S28E are found only in archaea and eukaryotes. Here we report the three‐dimensional solution structure of S28E by NMR spectroscopy. S28E contains a globular region and a long C‐terminal tail protruding from the core. The globular region consists of four antiparallel β‐strands that are arranged in a Greek‐key topology. Unique features of S28E include an extended loop L2–3 that folds back onto the protein and a 12‐residue charged C‐terminal tail with no regular secondary structure and greater flexibility relative to the rest of the protein. The structural and surface resemblance to OB‐fold family of proteins and the presence of highly conserved basic residues suggest that S28E may bind to RNA. A broad positively charged surface extending over one side of the β‐barrel and into the flexible C terminus may present a putative binding site for RNA.

Structural Similarity of YbeD Protein from Escherichia coli to Allosteric Regulatory Domains

Lipoic acid is an essential prosthetic group in several metabolic pathways. The biosynthetic pathway of protein lipoylation in Escherichia coli involves gene products of the lip operon. YbeD is a conserved bacterial protein located in the dacA-lipB intergenic region. Here, we report the nuclear magnetic resonance structure of YbeD from E. coli. The structure includes a beta alpha beta beta alpha beta fold with two alpha-helices on one side of a four-strand antiparallel beta-sheet. The beta 2-beta 3 loop shows the highest sequence conservation and is likely functionally important. The beta-sheet surface contains a patch of conserved hydrophobic residues, suggesting a role in protein-protein interactions. YbeD shows striking structural homology to the regulatory domain from d-3-phosphoglycerate dehydrogenase, hinting at a role in the allosteric regulation of lipoic acid biosynthesis or the glycine cleavage system.

Solution structure of ribosomal protein L40E, a unique C4 zinc finger protein encoded by archaeon Sulfolobus solfataricus

The ribosomal protein L40E from archaeon Sulfolobus solfataricus is a component of the 50S ribosomal subunit. L40E is a 56‐residue, highly basic protein that contains a C4 zinc finger motif, CRKC_X10_CRRC. Homologs are found in both archaea and eukaryotes but are not present in bacteria. Eukaryotic genomes encode L40E as a ubiquitin‐fusion protein. L40E was absent from the crystal structure of euryarchaeota 50S ribosomal subunit. Here we report the three‐dimensional solution structure of L40E by NMR spectroscopy. The structure of L40E is a three‐stranded β‐sheet with a simple β2β1β3 topology. There are two unique characteristics revealed by the structure. First, a large and ordered β2–β3 loop twists to pack across the one side of the protein. L40E contains a buried polar cluster comprising Lys19, Lys20, Cys22, Asn29, and Cys36. Second, the surface of L40E is almost entirely positively charged. Ten conserved basic residues are positioned on the two sides of the surface. It is likely that binding of zinc is essential in stabilizing the tertiary structure of L40E to act as a scaffold to create a broad positively charged surface for RNA and/or protein recognition.

Structural Analysis of HopPmaL Reveals the Presence of a Second Adaptor Domain Common to the HopAB Family of Pseudomonas syringae Type III Effectors

HopPmaL is a member of the HopAB family of type III effectors present in the phytopathogen Pseudomonas syringae. Using both X-ray crystallography and solution nuclear magnetic resonance, we demonstrate that HopPmaL contains two structurally homologous yet functionally distinct domains. The N-terminal domain corresponds to the previously described Pto-binding domain, while the previously uncharacterised C-terminal domain spans residues 308-385. While structurally similar, these domains do not share significant sequence similarity and most importantly demonstrate significant differences in key residues involved in host protein recognition, suggesting that each of them targets a different host protein.