Shoba Ranganathan

The whey acidic protein family: a new signature motif and three-dimensional structure by comparative modeling

Whey acidic proteins (WAP) from the mouse, rat, rabbit, camel, and pig comprise two four-disulfide core domains. From a detailed analysis of all sequences containing this domain, we propose a new PROSITE motif ([KRHGVLN]-X-¿PF¿-X-[CF]-[PQSVLI]-X(9,19)-C-¿P¿-X-[DN]-X-¿N¿ -[CE]-X(5)-C-C) to accurately identify new four-disulfide core proteins. A consensus model for the WAP proteins is proposed, based on the human mucous proteinase inhibitor crystal structure. This article presents a detailed atomic model for the two-domain porcine WAP sequence by comparative modeling. Surface electrostatic potential calculations indicate that the second domain of the pig WAP model is similar to the functional human mucous proteinase inhibitor domains, whereas the first domain may be nonfunctional.

kappa-Hefutoxin1, a novel toxin from the scorpion Heterometrus fulvipes with unique structure and function. Importance of the functional diad in potassium channel selectivity

An important and exciting challenge in the postgenomic era is to understand the functions of newly discovered proteins based on their structures. The main thrust is to find the common structural motifs that contribute to specific functions. Using this premise, here we report the purification, solution NMR, and functional characterization of a novel class of weak potassium channel toxins from the venom of the scorpion Heterometrus fulvipes. These toxins, κ-hefutoxin1 and κ-hefutoxin2, exhibit no homology to any known toxins. NMR studies indicate that κ-hefutoxin1 adopts a unique three-dimensional fold of two parallel helices linked by two disulfide bridges without any β−sheets. Based on the presence of the functional diad (Tyr5/Lys19) at a distance (6.0 ± 1.0 Å) comparable with other potassium channel toxins, we hypothesized its function as a potassium channel toxin. κ-Hefutoxin 1 not only blocks the voltage-gated K+-channels, Kv1.3 and Kv1.2, but also slows the activation kinetics of Kv1.3 currents, a novel feature of κ-hefutoxin 1, unlike other scorpion toxins, which are considered solely pore blockers. Alanine mutants (Y5A, K19A, and Y5A/K19A) failed to block the channels, indicating the importance of the functional diad.

The Gene for a Novel Member of the Whey Acidic Protein Family Encodes Three Four-disulfide Core Domains and Is Asynchronously Expressed during Lactation

Secretion of whey acidic protein (WAP) in milk throughout lactation has previously been reported for a limited number of species, including the mouse, rat, rabbit, camel, and pig. We report here the isolation of WAP from the milk of a marsupial, the tammar wallaby (Macropus eugenii). Tammar WAP (tWAP) was isolated by reverse-phase HPLC and migrates in SDS-polyacrylamide gel electrophoresis at 29.9 kDa. tWAP is the major whey protein, but in contrast to eutherians, secretion is asynchronous and occurs only from approximately days 130 through 240 of lactation. The full-length cDNA codes for a mature protein of 191 amino acids, which is comprised of three four-disulfide core domains, contrasting with the two four-disulfide core domain arrangement in all other known WAPs. A three-dimensional model for tWAP has been constructed and suggests that the three domains have little interaction and could function independently. Analysis of the amino acid sequence suggests the protein belongs to a family of protease inhibitors; however, the predicted active site of these domains is dissimilar to the confirmed active site for known protease inhibitors. This suggests that any putative protease ligand may be unique to either the mammary gland, milk, or gut of the pouch young. Examination of the endocrine regulation of thetWAP gene showed consistently that the gene is prolactin-responsive but that the endocrine requirements for induction and maintenance of tWAP gene expression are different during lactation.

Multiple ligand binding sites on domain seven of human complement factor H

Foreign particles and damaged host cells can activate the complement system leading to their destruction by the host defense system. Factor H (fH) plays a vital role in restricting complement activation on host cells through interactions with polyanions such as heparin, while allowing activation to proceed on foreign surfaces. Complement activation by damaged host cells is also down regulated by fH, which is localized to injured areas through interactions with C-reactive protein (CRP). A number of pathogens have developed mechanisms by which they can also bind fH and thus exploit its protective properties. One such organism is Group A Streptococcus (GAS) which mediates fH binding via its surface expressed M-protein. fH consists of 20 conserved short consensus repeat (SCR) units and mutagenesis studies indicate that the seventh repeat is responsible for interactions with heparin, CRP and M-protein. We recently performed molecular modelling of fH SCR 7 and identified a cluster of positively charged residues on one face of the domain. By alanine replacement mutagenesis, we demonstrated that these residues are involved in heparin, CRP and M protein binding, which indicates that there is a common site within fH SCR 7 responsible for multiple ligand recognition.

Complement factor H: sequence analysis of 221 kb of human genomic DNA containing the entire fH, fHR-1 and fHR-3 genes.

Complement factor H (fH) is a member of a family of proteins involved in the regulation of complement activation (RCA). These proteins share a common structural motif, the Short Consensus Repeat (SCR), which is structurally conserved among related genes and between phylogenetically divergent species. fH is composed of 20 such SCRs and a variety of biological functions have been localised to specific SCR domains. The majority of individual SCRs identified are encoded by single exons, and processes such as gene conversion, duplication and exon shuffling have been implicated in the evolution and genomic radiation of SCR-encoding genes. We have analysed two GenBank sequence entries relating to two overlapping PAC clones sequenced at the Sanger Centre which contain the entire human fH gene and two adjacent fH-related (fHR) genes, fHR-1 and fHR-3. Here, we report the detailed analysis of the assembled 221 kb of contiguous, ungapped genomic sequence from human chromosome 1q32, in part employing the RUMMAGE-DP automated annotation tool. Genomic duplications involving fH and fHR exons were identified and Alu/L1 repeat dating established that the duplications occurred after the separation of rodent and primate lineages. The analysis indicates that retrotransposition as well as single and multiple exon duplication events are likely to have been involved in SCR radiation and RCA gene evolution, facilitated by conservation of splice-phasing and the single-exon, single-SCR nature of the encoded domains.

Mechanistic aspects of biological redox reactions involving NADH. Part 5.—AM1 transition-state studies for the pyruvate–L-lactate interconversion in L-lactate dehydrogenase

The catalytic mechanism for the interconversion of pyruvate to L-lactate by the enzyme L-lactate dehydrogenase (LDH), in the presence of the cofactor nicotinamide adenine dinucleotide (NAD), has been studied using semiempirical AM1 quantum mechanical calculations. We have characterized the structure of the LDH transition state (TS), in isolation and in the presence of key active-site groups, using a supermolecule model. An initial investigation with isolated substrate and cofactor analogues resulted in TS structures for hydride-ion transfer from the cofactor analogue, planar trans-1- methyldihydronicotinamide to eight conformers of the substrate analogue, protonated pyruvic acid. Fragments of essential active-site residues were then introduced in stages. With truncated Arg-171 and His-195 residues, the TS for hydride transfer from the cofactor analogue to the substrate pyruvate resembled the active-site configuration in the X-ray crystallographic structure of the abortive LDH–NADH–oxamate ternary complex. The substrate species is carbonyl-protonated and thus the rate-limiting chemical step is hydride transfer. These results contrast with earlier work indicating that carbonyl-protonated pyruvate is unstable in the free state (K. E. Norris, G. B. Bacskay and J. E. Gready, J. Comput. Chem., 1993, 14, 699). Introduction of the Val-138 fragment gave closer agreement with experiment for the orientation of the cofactor analogues carboxamide side chain in the TS and for the reversibility criteria for the reaction. For each TS located, stable reactant and product complexes have been isolated by following the reaction coordinate, and the optimized structures, energies and charge distributions of the TS, stable reactant and product complexes and the isolated reactants and products are reported. There is significant charge transfer in the TS, with a charge of ca.+ 0.4 on the nicotinamide species.

Pinpointing the putative heparin/sialic acid-binding residues in the 'sushi' domain 7 of factor H: a molecular modeling study.

Factor H, a secretory glycoprotein comprising 20 short consensus repeat (SCR) or sushi domains of about 60 amino acids each, is a regulator of the complement system. The complement-regulatory functions of factor H are targeted by its binding to polyanions such as heparin/sialic acid, involving SCRs 7 and 20. Recently, the SCR 7 heparin-binding site was shown to be co-localized with the Streptococcus Group A M protein binding site on factor H (T.K. Blackmore et al., Infect. Immun. 66, 1427 (1998)). Using sequence analysis of all heparin-binding domains of factor H and its closest homologues, molecular modeling of SCRs 6 and 7, and surface electrostatic potential studies, the residues implicated in heparin/sialic acid binding to SCR 7 have been localized to four regions of sequence space containing stretches of basic as well as histidine residues. The heparin-binding site is spatially compact and lies near the interface between SCRs 6 and 7, with residues in the interdomain linker playing a significant role.

A common site within factor H SCR 7 responsible for binding heparin, C-reactive protein and streptococcal M protein

The complement inhibitor factor H (fH) interacts via its seventh short consensus repeat (SCR) domain with multiple ligands including heparin, streptococcal M protein and C-reactive protein (CRP). The aim of this study was to localize the residues in SCR 7 required for these interactions. We initially built a homology model of fH SCR 6-7 using the averaged NMR structures of fH SCR 15-16 and vaccinia control protein SCR 3-4 as templates. Electrostatic potentials of the models surface demonstrated a co-localization of three clusters of positively charged residues on SCR 7, labeled site A (R369 and K370), site B (R386 and K387) and site C (K392). These residues, localized to the linker region preceding SCR 7 and to the end of a hypervariable loop in SCR 7, were systematically replaced with uncharged alanine residues in an fH construct containing SCR 1-7. The resulting proteins were expressed in the methylotrophic yeast, Pichia pastoris. By ELISA analysis we demonstrated: first, that substituting site A inhibited heparin and CRP binding; secondly, that substituting site B inhibited binding to heparin, CRP and M protein; and thirdly, that substituting site C clearly inhibited only heparin binding.