Andrew C. R. Martin

Bacterial Virulence in the Moonlight: Multitasking Bacterial Moonlighting Proteins Are Virulence Determinants in Infectious Disease

ABSTRACT Men may not be able to multitask, but it is emerging that proteins can. This capacity of proteins to exhibit more than one function is termed protein moonlighting, and, surprisingly, many highly conserved proteins involved in metabolic regulation or the cell stress response have a range of additional biological actions which are involved in bacterial virulence. This review highlights the multiple roles exhibited by a range of bacterial proteins, such as glycolytic and other metabolic enzymes and molecular chaperones, and the role that such moonlighting activity plays in the virulence characteristics of a number of important human pathogens, including Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Helicobacter pylori, and Mycobacterium tuberculosis.

Association between human rhinovirus C and severity of acute asthma in children

A new and potentially more pathogenic group of human rhinovirus (HRV), group C (HRVC), has recently been discovered. We hypothesised that HRVC would be present in children with acute asthma and cause more severe attacks than other viruses or HRV groups. Children with acute asthma (n = 128; age 2–16 yrs) were recruited on presentation to an emergency department. Asthma exacerbation severity was assessed, and respiratory viruses and HRV strains were identified in a nasal aspirate. The majority of the children studied had moderate-to-severe asthma (85.2%) and 98.9% were admitted to hospital. HRV was detected in 87.5% and other respiratory viruses in 14.8% of children, most of whom also had HRV. HRVC was present in the majority of children with acute asthma (59.4%) and associated with more severe asthma. Children with HRVC (n = 76) had higher asthma severity scores than children whose HRV infection was HRVA or HRVB only (n = 34; p = 0.018), and all other children (n = 50; p = 0.016). Of the 19 children with a non-HRV virus, 13 had HRV co-infections, seven of these being HRVC. HRVC accounts for the majority of asthma attacks in children presenting to hospital and causes more severe attacks than previously known HRV groups and other viruses.

Analysis of the antigen combining site: Correlations between length and sequence composition of the hypervariable loops and the nature of the antigen

It has long been suggested that the overall shape of the antigen combining site (ACS) of antibodies is correlated with the nature of the antigen. For example, deep pockets are characteristic of antibodies that bind haptens, grooves indicate peptide binders, while antibodies that bind to proteins have relatively flat combining sites. In 1996, MacCallum, Martin and Thornton used a fractal shape descriptor and showed a strong correlation of the shape of the binding region with the general nature of the antigen.However, the shape of the ACS is determined primarily by the lengths of the six complementarity-determining regions (CDRs). Here, we make a direct correlation between the lengths of the CDRs and the nature of the antigen. In addition, we show significant differences in the residue composition of the CDRs of antibodies that bind to different antigen classes. As well as helping us to understand the process of antigen recognition, autoimmune disease and cross-reactivity, these results are of direct application in the design of antibody phage libraries and modification of affinity.

Protein folds and functions

BACKGROUND The recent rapid increase in the number of available three-dimensional protein structures has further highlighted the necessity to understand the relationship between biological function and structure. Using structural classification schemes such as SCOP, CATH and DALI, it is now possible to explore global relationships between protein fold and function, something which was previously impractical. RESULTS Using a relational database of CATH data we have generated fold distributions for arbitrary selections of proteins automatically. These distributions have been examined in the light of protein function and bound ligand. Different enzyme classes are not clearly reflected in distributions of protein class and architecture, whereas the type of bound ligand has a much more dramatic effect. CONCLUSIONS The availability of structural classification data has enabled this novel overview analysis. We conclude that function at the top level of the EC number enzyme classification is not related to fold, as only a very few specific residues are actually responsible for enzyme activity. Conversely, the fold is much more closely related to ligand type.

Accessing the Kabat antibody sequence database by computer

The Kabat antibody sequence database has for many years been the primary site for depositing sequence information on antibodies and other proteins of immunological interest. The chief drawback of this database has been that it has only been available in the form of a printed book (Kabat et al., Sequences of Proteins of Immunological Interest, 1991). These data have recently become available on the global computer Internet, but no method of searching the data has, as yet, been provided. Here, the development of a specialized database program for accessing the antibody data is described. This database software has been made accessible over the World Wide Web, together with a program which allows a novel antibody sequence to be tested against the Kabat sequence database, to identify unusual features of an antibody sequence which may represent cloning artifacts or sequencing errors.

Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains

In analysing protein sequence and structure, standardized numbering schemes allow comparison of features without explicit alignment. This has proved particularly valuable in the case of antibodies. The most widely used schemes (Kabat: sequence-based; Chothia: structure-based) differ only in the numbering of the complementarity determining regions (CDRs). We have analyzed the numbered annotations in the widely used Kabat database and found that approximately 10% of entries contain errors or inconsistencies. Further analysis of sequence alignments in the context of structure suggest that the sites of the insertions in some framework regions in the Kabat and Chothia schemes are incorrect. We therefore propose a corrected version of the Chothia scheme which is structurally correct throughout the CDRs and frameworks. To perform this analysis, we have developed, and made available, a tool for the automatic application of Kabat, Chothia and modified-Chothia numbering schemes and have carefully benchmarked the performance of this tool.

Spt4/5 stimulates transcription elongation through the RNA polymerase clamp coiled-coil motif

Spt5 is the only known RNA polymerase-associated factor that is conserved in all three domains of life. We have solved the structure of the Methanococcus jannaschii Spt4/5 complex by X-ray crystallography, and characterized its function and interaction with the archaeal RNAP in a wholly recombinant in vitro transcription system. Archaeal Spt4 and Spt5 form a stable complex that associates with RNAP independently of the DNA–RNA scaffold of the elongation complex. The association of Spt4/5 with RNAP results in a stimulation of transcription processivity, both in the absence and the presence of the non-template strand. A domain deletion analysis reveals the molecular anatomy of Spt4/5—the Spt5 Nus-G N-terminal (NGN) domain is the effector domain of the complex that both mediates the interaction with RNAP and is essential for its elongation activity. Using a mutagenesis approach, we have identified a hydrophobic pocket on the Spt5 NGN domain as binding site for RNAP, and reciprocally the RNAP clamp coiled-coil motif as binding site for Spt4/5.

Assessment of comparative modeling in CASP2

An assessment is presented for all submissions to the comparative modeling challenge in the 1996 Critical Assessment of Structure Prediction (CASP2). Of the original 12 target structures, 9 were solved prior to the meeting: 8 by X‐ray crystallography and 1 by NMR spectroscopy. These targets varied over a large range of difficulty, as assessed by the percentage sequence identity with the principal parent structure, which ranged from 20% up to 85%. The overall quality of the models reflected the similarity of the principal parent. As expected, when the sequence alignment was correct, the core was accurately modeled, with the largest deviations occurring in the loops. Models were built which gave Cα root‐mean‐square deviations (RMSDs) compared with the observed structure of <1 Å for targets with high parental similarity; even at 26% sequence identity, the best model structures had Cα deviations of only 2.2 Å. Overall, these deviations are comparable with those observed between the parent structure and the target, but locally there are several examples where the model approaches closer to the target than does the parent. There were three targets below 25% sequence identity, and the models generated for these targets were, in general, significantly less accurate. This principally reflects errors in the alignment which, if systematically shifted, can generate Cα RMSDs <18 Å. Compared with CASP1, the geometry of the models was significantly improved with no D‐amino acids. By far the major contribution to RMSD error was the alignment accuracy, which varied from 100% down to 7% over the range of targets. In the structurally variable regions, global shifts, caused by hinge bending, were the major source of error, giving significantly lower local RMSDs than global RMSDs. In over 50% of these noncore regions, the difference between global and local RMSDs was more than 3 Å, and was as high as 10 Å for one structurally variable region. For the side chains, the χ1 RMSDs are strongly correlated with the Cα RMSDs. For models with Cα deviations less than 1 Å, on average 78.5% of side chains are placed in the correct rotamer, although the χ1 RMSDs, though clearly better than random, were disappointing at around 46°. As the backbone deviations increased, the side chain placement became less accurate, with an average χ1 RMSD of 75° on a 1.5–2.5 Å Cα backbone (average 51.4% correct rotamer). Refinement by energy minimization or molecular dynamics made only minor adjustments to improve local geometry and generally made small, but not significant, improvements to the RMSD. In total, 19 groups submitted 62 models (89 coordinate sets) that could be assessed. Most modelers used manual adjustments to sequence alignments and, in general, good alignments were obtained down to 25% sequence identity. The modeling methods ranged from “classical” modeling, involving core building followed by loop and side chain addition, to more sophisticated approaches based on probability distributions, Monte Carlo sampling or distance geometry. For each target, several groups produced equally good models, given the expected errors in the structures (about 0.5 Å). No one method came out as clearly superior, although the approaches that inherit directly from the parents generally performed better than the more radical techniques. However, for each target there were some poor models, usually reflecting a poor sequence alignment, and the range of accuracy for each target is therefore large. Fully automated methods are able to perform very well for “easy” targets (85% sequence identity with parent), but when modeling using a distantly related parent, care and expertise, especially in performing the alignment, still appear to be important factors in generating accurate models. Proteins, Suppl. 1:14–28, 1997.

Genetic, structural and functional properties of an IgG DNA-binding monoclonal antibody from a lupus patient with nephritis

Antibodies binding to double‐stranded (ds) DNA are strongly associated with renal involvement in patients with systemic lupus erythematosus (SLE). We have generated two new IgG DNA‐binding monoclonal antibodies (mAb), RH‐14 and DIL‐6, from the peripheral blood lymphocytes of two SLE patients with glomerulonephritis using the heteromyeloma cell line CB‐F7. RH‐14 is an IgG1 λ antibody which also bound to single‐stranded (ss)DNA, histones and nucleosomes. DIL‐6 is an IgG3 λ antibody with restricted antigen binding specificity. cDNA encoding the variable regions of the heavy (VH) and light (VL) chains of RH‐14 was sequenced and the antigen binding site of this mAb was computer modelled. Sequence analysis of VH and VL regions of RH‐14 showed that VH is derived from germ‐line gene V3‐7, a member of the VH3 family, and VL is derived from DPL 11, a member of the Vλ2 family. Somatic mutations and basic amino acid residues are identified in the complementarity‐ determining regions of both VH and VL regions. The nephritogenic properties of these mAb were analyzed by implanting and growing the hybridoma cells secreting the mAb in the peri toneum of SCID mice. The animals that received the RH‐14 hybridoma produced higher levels of proteinuria (3 to ≥ 4) (p < 0.001) compared to the groups that received DIL‐6 (trace to ≥ 1) or CB‐F7 (trace). Electron microscopy of kidney sections from all the RH‐14‐ implanted animals showed granular immunoglobulin deposition in the renal glomerular capil laries and mesangium. In this study we have shown for the first time using electron micros copy that a human IgG anti‐dsDNA mAb, RH‐14, is nephritogenic and that deposition of such an antibody alone is sufficient to induce renal damage.

Bacterial moonlighting proteins and bacterial virulence.

Implicit in the central dogma is the hypothesis that each protein gene product has but one function. However, over the past decade, it has become clear that many proteins have one or more unique functions, over-and-above the principal biological action of the specific protein. This phenomenon is now known as protein moonlighting and many well-known proteins such as metabolic enzymes and molecular chaperones are now recognised as moonlighting proteins. A growing number of bacterial species are being found to have moonlighting proteins and the moonlighting activities of such proteins can contribute to bacterial virulence behaviour. The glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPD) and enolase, and the cell stress proteins: chaperonin 60, Hsp70 and peptidyl prolyl isomerase, are among the most common of the bacterial moonlighting proteins which play a role in bacterial virulence. Moonlighting activities include adhesion and modulation of cell signalling processes. It is likely that only the tip of the bacterial moonlighting iceberg has been sighted and the next decade will bring with it many new discoveries of bacterial moonlighting proteins with a role in bacterial virulence.