Adelinda Yee

Structural proteomics of an archaeon.

A set of 424 nonmembrane proteins from Methanobacterium thermoautotrophicum were cloned, expressed and purified for structural studies. Of these, ∼20% were found to be suitable candidates for X-ray crystallographic or NMR spectroscopic analysis without further optimization of conditions, providing an estimate of the number of the most accessible structural targets in the proteome. A retrospective analysis of the experimental behavior of these proteins suggested some simple relations between sequence and solubility, implying that data bases of protein properties will be useful in optimizing high throughput strategies. Of the first 10 structures determined, several provided clues to biochemical functions that were not detectable from sequence analysis, and in many cases these putative functions could be readily confirmed by biochemical methods. This demonstrates that structural proteomics is feasible and can play a central role in functional genomics.

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.

In situ proteolysis for protein crystallization and structure determination

We tested the general applicability of in situ proteolysis to form protein crystals suitable for structure determination by adding a protease (chymotrypsin or trypsin) digestion step to crystallization trials of 55 bacterial and 14 human proteins that had proven recalcitrant to our best efforts at crystallization or structure determination. This is a work in progress; so far we determined structures of 9 bacterial proteins and the human aminoimidazole ribonucleotide synthetase (AIRS) domain.

SPINE: an integrated tracking database and data mining approach for identifying feasible targets in high-throughput structural proteomics

High-throughput structural proteomics is expected to generate considerable amounts of data on the progress of structure determination for many proteins. For each protein this includes information about cloning, expression, purification, biophysical characterization and structure determination via NMR spectroscopy or X-ray crystallography. It will be essential to develop specifications and ontologies for standardizing this information to make it amenable to retrospective analysis. To this end we created the SPINE database and analysis system for the Northeast Structural Genomics Consortium. SPINE, which is available at bioinfo.mbb.yale.edu/nesg or nesg.org, is specifically designed to enable distributed scientific collaboration via the Internet. It was designed not just as an information repository but as an active vehicle to standardize proteomics data in a form that would enable systematic data mining. The system features an intuitive user interface for interactive retrieval and modification of expression construct data, query forms designed to track global project progress and external links to many other resources. Currently the database contains experimental data on 985 constructs, of which 740 are drawn from Methanobacterium thermoautotrophicum, 123 from Saccharomyces cerevisiae, 93 from Caenorhabditis elegans and the remainder from other organisms. We developed a comprehensive set of data mining features for each protein, including several related to experimental progress (e.g. expression level, solubility and crystallization) and 42 based on the underlying protein sequence (e.g. amino acid composition, secondary structure and occurrence of low complexity regions). We demonstrate in detail the application of a particular machine learning approach, decision trees, to the tasks of predicting a proteins solubility and propensity to crystallize based on sequence features. We are able to extract a number of key rules from our trees, in particular that soluble proteins tend to have significantly more acidic residues and fewer hydrophobic stretches than insoluble ones. One of the characteristics of proteomics data sets, currently and in the foreseeable future, is their intermediate size ( approximately 500-5000 data points). This creates a number of issues in relation to error estimation. Initially we estimate the overall error in our trees based on standard cross-validation. However, this leaves out a significant fraction of the data in model construction and does not give error estimates on individual rules. Therefore, we present alternative methods to estimate the error in particular rules.

Structural proteomics: prospects for high throughput sample preparation

1. BackgroundWith the near completion of many genome sequencing projects has come the soberingrealisation that our understanding of biology is nowhere near complete. For example, inthe worm, C. elegans, less than half of the predicted proteins have a known function(Consortium, 1998). The major challenge facing biologists in the next decade will be to‘‘finish the job’’, that is, to ascribe a function to each of the proteins that have been discovered

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.

1HC and 1HN total NOE correlations in a single 3D NMR experiment. 15N and 13C time-sharing in t1 and t2 dimensions for simultaneous data acquisition

Simultaneous data acquisition in time-sharing (TS) multi-dimensional NMR experiments has been shown an effective means to reduce experimental time, and thus to accelerate structure determination of proteins. This has been accomplished by spin evolution time-sharing of the X and Y heteronuclei, such as 15N and 13C, in one of the time dimensions. In this work, we report a new 3D TS experiment, which allows simultaneous 13C and 15N spin labeling coherence in both t1 and t2 dimensions to give four NOESY spectra in a single 3D experiment. These spectra represent total NOE correlations between 1HN and 1HC resonances. This strategy of double time-sharing (2TS) results in an overall four-fold reduction in experimental time compared with its conventional counterpart. This 3D 2TS CN-CN-H HSQC-NOESY-HSQC pulse sequence also demonstrates improvements in water suppression, 15N spectral resolution and sensitivity, which were developed based on 2D TS CN-H HSQC and 3D TS H-CN-H NOESY-HSQC experiments. Combining the 3D TS and the 3D 2TS NOESY experiments, NOE assignment ambiguities and errors are considerably reduced. These results will be useful for rapid protein structure determination to complement the effort of discerning the functions of diverse genomic proteins.

NMR solution structure of Thermotoga maritima protein TM1509 reveals a Zn-metalloprotease-like tertiary structure.

The 150-residue protein TM1509 is encoded in gene YF09_THEMA of Thermotoga maritima. TM1509 has so far no functional annotation and belongs to protein family UPF0054 (PFAM accession number: PF02130) which contains at least 146 members. The NMR structure of TM1509 reveals an α+β fold comprising a four stranded β-sheet with topology A(↑), B(↑), D(↑), C(↓) as well as five α-helices I–V. The structures of most members of family PF02130 can be reliably constructed using the TM1509 NMR structure, demonstrating high leverage for exploration of fold space. A multiple sequence alignment of TM1509 with homologues of family UPF0054 shows that three polypeptide segments, as well as a putative zinc-binding consensus motif HGXLHLXGYDH located at the C-terminal end of α-helix IV, are highly conserved. The spatial arrangement of the three His residues of this UPF0054 consensus motif is similar to the arrangement found for the His residues in the HEXXHXXGXXH zinc-binding consensus motif of matrix metallo-proteases (MMPs). Moreover, the other conserved polypeptide segments form a large cavity which encloses the putative Zn-binding pocket and might confer specificity during catalysis. However, TM1509 and the other members of the UPF0054 family do not have the crucial Glu residue in position 2 of the MMP consensus motif. Intriguingly, the TM1509 structure indicates that the Asp in the UPF0054 consensus motif (Asp 111 in TM1509) may overtake the catalytic role of the Glu. This suggests that protein family UPF0054 might contain members of a hitherto uncharacterized class of metalloproteases.