Structural and functional consequences of succinate dehydrogenase subunit B mutations.

Mitochondrial dysfunction, due to mutations of the gene encoding succinate dehydrogenase (SDH), has been implicated in the development of adrenal phaeochromocytomas, sympathetic and parasympathetic paragangliomas, renal cell carcinomas, gastrointestinal stromal tumours and more recently pituitary tumours. Underlying mechanisms behind germline SDH subunit B (SDHB) mutations and their associated risk of disease are not clear. To investigate genotype-phenotype correlation of SDH subunit B (SDHB) variants, a homology model for human SDH was developed from a crystallographic structure. SDHB mutations were mapped, and biochemical effects of these mutations were predicted in silico. Results of structural modelling indicated that many mutations within SDHB are predicted to cause either failure of functional SDHB expression (p.Arg27*, p.Arg90*, c.88delC and c.311delAinsGG), or disruption of the electron path (p.Cys101Tyr, p.Pro197Arg and p.Arg242His). GFP-tagged WT SDHB and mutant SDHB constructs were transfected (HEK293) to determine biological outcomes of these mutants in vitro. According to in silico predictions, specific SDHB mutations resulted in impaired mitochondrial localisation and/or SDH enzymatic activity. These results indicated strong genotype-functional correlation for SDHB variants. This study reveals new insights into the effects of SDHB mutations and the power of structural modelling in predicting biological consequences. We predict that our functional assessment of SDHB mutations will serve to better define specific consequences for SDH activity as well as to provide a much needed assay to distinguish pathogenic mutations from benign variants.

Molecular modelling and site-directed mutagenesis of human GALR1 galanin receptor defines determinants of receptor subtype specificity.

Human galanin is a 30 amino acid neuropeptide that elicits a range of biological activities by interaction with G protein-coupled receptors. We have generated a model of the human GALR1 galanin receptor subtype (hGALR1) based on the alpha carbon maps of frog rhodopsin and investigated the significance of potential contact residues suggested by the model using site-directed mutagenesis. Mutation of Phe186 within the second extracellular loop to Ala resulted in a 6-fold decrease in affinity for galanin, representing a change in free energy consistent with hydrophobic interaction. Our model suggests interaction between Phe186 of hGALR1 and Ala7 or Leu11 of galanin. Receptor subtype specificity was investigated by replacement of residues in hGALR1 with the corresponding residues in hGALR2 and use of the hGALR2-specific ligands hGalanin(2-30) and [D-Trp2]hGalanin(1-30). The His267Ile mutant receptor exhibited a pharmacological profile corresponding to that of hGALR1, suggesting that His267 is not involved in a receptor-ligand interaction. The mutation Phe115Ala resulted in a decreased binding affinity for hGalanin and for hGALR2-specific analogues, indicating Phe115 to be of structural importance to the ligand binding pocket of hGALR1 but not involved in direct ligand interaction. Analysis of Glu271Trp suggested that Glu271 of hGALR1 interacts with the N-terminus of galanin and that the Trp residue in the corresponding position in hGALR2 is involved in receptor subtype specificity of binding. Our model supports previous reports of Phe282 of hGALR1 interacting with Trp2 of galanin and His264 of hGALR1 interacting with Tyr9 of galanin.

Modeling of the structural features of integral-membrane proteins reverse-environment prediction of integral membrane protein structure (REPIMPS).

The Profiles-3D application, an inverse-folding methodology appropriate for water-soluble proteins, has been modified to allow the determination of structural properties of integral-membrane proteins (IMPs) and for testing the validity of solved and model structures of IMPs. The modification, known as reverse-environment prediction of integral membrane protein structure (REPIMPS), takes into account the fact that exposed areas of side chains for many residues in IMPs are in contact with lipid and not the aqueous phase. This (1) allows lipid-exposed residues to be classified into the correct physicochemical environment class, (2) significantly improves compatibility scores for IMPs whose structures have been solved, and (3) reduces the possibility of rejecting a three-dimensional structure for an IMP because the presence of lipid was not included. Validation tests of REPIMPS showed that it (1) can locate the transmembrane domain of IMPs with single transmembrane helices more frequently than a range of other methodologies, (2) can rotationally orient transmembrane helices with respect to the lipid environment and surrounding helices in IMPs with multiple transmembrane helices, and (3) has the potential to accurately locate transmembrane domains in IMPs with multiple transmembrane helices. We conclude that correcting for the presence of the lipid environment surrounding the transmembrane segments of IMPs is an essential step for reasonable modeling and verification of the three-dimensional structures of these proteins.

A novel approach to the design of inhibitors of human secreted phospholipase A2 based on native peptide inhibition.

Human Type IIA secreted phospholipase A(2) (sPLA(2)-IIA) is an important modulator of cytokine-dependent inflammatory responses and a member of a growing superfamily of structurally related phospholipases. We have previously shown that sPLA(2)-IIA is inhibited by a pentapeptide sequence comprising residues 70-74 of the native sPLA(2)-IIA protein and that peptides derived from the equivalent region of different sPLA(2)-IIA species specifically inhibit the enzyme from which they are derived. We have now used an analogue screen of the human pentapeptide (70)FLSYK(74) in which side-chain residues were substituted, together with molecular docking approaches that modeled low-energy conformations of (70)FLSYK(74) bound to human sPLA(2)-IIA, to generate inhibitors with improved potency. Importantly, the modeling studies showed a close association between the NH(2) and COOH termini of the peptide, predicting significant enhancement of the potency of inhibition by cyclization. Cyclic compounds were synthesized and indeed showed 5-50-fold increased potency over the linear peptide in an Escherichia coli membrane assay. Furthermore, the potency of inhibition correlated with steady-state binding of the cyclic peptides to sPLA(2)-IIA as determined by surface plasmon resonance studies. Two potential peptide interaction sites were identified on sPLA(2)-IIA from the modeling studies, one in the NH(2)-terminal helix and the other in the beta-wing region, and in vitro association assays support the potential for interaction of the peptides with these sites. The inhibitors were effective at nanomolar concentrations in blocking sPLA(2)-IIA-mediated amplification of cytokine-induced prostaglandin synthesis in human rheumatoid synoviocytes in culture. These studies provide an example where native peptide sequences can be used for the development of potent and selective inhibitors of enzyme function.

Crystallization and preliminary X-ray diffraction studies of a new crystal form of human secretory type IIA phospholipase A2.

Human synovial type IIA phospholipase A(2) (sPLA(2)-IIA) has been implicated in the pathogenesis of a number of inflammatory diseases and is a target for the development of therapeutically useful inhibitors. Biochemical evidence suggests a novel mechanism of inhibition for a series of peptide inhibitors originally derived from the primary sequence of the protein. On co-incubation with one of these inhibitors, single crystals of a hitherto unreported crystallographic form of sPLA2-IIA suitable for diffraction analysis were obtained. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 140.8, b = 38.9, c = 109.1 A, beta = 125.1 degrees, and diffraction at 2.4 A resolution has been observed.

Relating structure to function in the beta-propeller domain of dipeptidyl peptidase IV. Point mutations that influence adenosine deaminase binding, antibody binding and enzyme activity.

Point mutations in human CD26/DP IV were analysed for adenosine deaminase (ADA) binding, monoclonal antibody (mAb) binding and DP IV enzyme activity. Point mutations at either Leu294 or Val341 ablated ADA binding. Binding by mAbs that inhibit ADA binding was found to involve both Leu340 to Arg343 and Thr440/Lys441. Glu205 and Glu206 were found to be essential for enzyme activity. All residues of interest were mapped onto a model of the beta-propeller domain of DP IV. These data led us to suggest that in DP IV and related peptidases ligand and antibody binding sites are non-linear and that enzyme activity depends on charged sidechains that surround the entrance to the central tunnel of the beta-propeller.

Molecular pathogenesis of liver disease: an approach to hepatic inflammation, cirrhosis and liver transplant tolerance.

The hallmarks of chronic liver diseases are chronic inflammation, cellular damage, regeneration and fibrosis. An appreciation of intrahepatic molecular expression patterns in normal and diseased liver provides clues for understanding pathogenic pathways whilst studies of the structure and function of molecules implicated in liver disease provide insights into their potential as therapeutic targets. We have examined the expression, function, molecular structure and structure-function relationships of type IV dipeptidyl aminopeptidases. In particular, the roles of CD26/DPPIV in T-cell proliferation and chemotaxis and of fibroblast activation protein in human cirrhosis are discussed. We have investigated the pathogenesis of liver disease by characterising patterns of cytokine and growth factor expression in experimental and human cirrhosis. We have quite recently expanded this approach to use differential gene expression analyses to elucidate overall pathways of gene activation and suppression in human cirrhosis. In addition, our detailed molecular and cellular studies of the mechanisms of spontaneous liver transplant tolerance have generated novel insights into this process. This review touches on these diverse aspects of liver function and disease.

GTP binding and signaling by Gh/transglutaminase II involves distinct residues in a unique GTP-binding pocket.

G(h) is a dual function protein. It has receptor signaling activity that requires GTP binding and Ca(2+)-activated transglutaminase (TGase) activity that is inhibited by GTP binding. G(h) shows no homology with other GTP-binding proteins, and its GTP-binding site has not been defined. Based on sequence analysis of [alpha-(32)P]GTP-photolabeled and proteolytically released internal peptide fragments, we report localization of GTP binding to a 15-residue segment ((159)YVLTQQGFIYQGSVK(173)) of the G(h) core domain. This was confirmed by site-directed mutagenesis; a G(h)/fXIIIA chimera (in which residues 162-179 of G(h) were substituted with the equivalent but nonhomologous region of the non-GTP-binding TGase factor XIIIA) and a G(h) point mutant, S171E, retained TGase activity but failed to bind and hydrolyze GTP and did not support alpha(1B)-adrenergic receptor signaling. Slight impairment of GTP binding (1.5-fold) and hydrolysis (10-fold) in the absence of altered TGase activity did not affect signaling by the mutant K173N. However, greater impairment of GTP binding (6-fold) and hydrolysis (50-fold) abolished signaling by the mutant K173L. Mutant S171C exhibited enhanced GTP binding and signaling. Thus, residues Ser(171) and Lys(173) are critical for both GTP binding and signaling but not TGase activity. Mutagenesis of residues N-terminal to Gly(170) impaired both GTP binding and TGase activity. From computer modeling of G(h), it is evident that the GTP-binding region identified here is distinct from, but interacts with, the TGase active site. Together with structural considerations of G(h) versus other GTP-binding proteins, these findings indicate that G(h) has a unique GTP-binding pocket and provide for the first time a mechanism for GTP-mediated regulation of the TGase activity of G(h).

The function of conserved amino acid residues adjacent to the effector domain in elongation factor G.

Bacterial elongation factor G (EF-G) physically associates with translocation-competent ribosomes and facilitates transition to the subsequent codon through the coordinate binding and hydrolysis of GTP. In order to investigate the amino acid positions necessary for EF-G functions, a series of mutations were constructed in the EF-G structural gene (fusA) of Escherichia coli, specifically at positions flanking the effector domain. A mutated allele was isolated in which the wild-type sequence from codons 29 to 47 ("EFG2947") was replaced with a sequence encoding 28 amino acids from ribosomal protein S7. This mutated gene was unable to complement a fusAts strain when supplied in trans at the nonpermissive temperature. In vitro biochemical analysis demonstrated that nucleotide crosslinking was unaffected in EFG2947, while ribosome binding appeared to be completely abolished. A series of point mutations created within this region, encoding L30A, Y32A, H37A, and K38A were shown to give rise to fully functional proteins, suggesting that side chains of these individual residues are not essential for EF-G function. A sixth mutant, E41A, was found to inefficiently rescue growth in a fusAts background, and was also unable to bind ribosomes normally in vitro. In contrast E41Q could restore growth at the nonpermissive temperature. These results can be explained within the context of a three-dimensional model for the effector region of EF-G. This model indicates that the effector domain contains a negative potential field that may be important for ribosome binding.

Binding to human dipeptidyl peptidase IV by adenosine deaminase and antibodies that inhibit ligand binding involves overlapping, discontinuous sites on a predicted beta propeller domain.

Dipeptidyl peptidase IV (DPPIV) is an atypical serine protease that modifies the biological activities of certain chemokines and neuropeptides. In addition, human DPPIV, also known as the T-cell activation antigen CD26, binds adenosine deaminase (ADA) to the T-cell surface, thus protecting the T-cell from adenosine-mediated inhibition of proliferation. Mutations were engineered into DPPIV (five point, 16 single point and six deletion mutations) to examine the binding of ADA and 19 monoclonal antibodies. Deletions of C-terminal residues from the 738-residue extracellular portion of DPPIV showed that the 214 residues C-terminal to Ser552 were not required for ADA binding and that peptidase activity could be ablated by deletion of 20 residues from the C-terminus. Point mutations at either of two locations, Leu294 and Val341, ablated ADA binding. Binding by six anti-DPPIV antibodies that inhibited ADA binding was found to require Leu340 to Arg343 and Thr440/Lys441 but not the 214 residues C-terminal to Ser552. The 13 other antibodies studied bound to a truncated DPPIV consisting of amino acids 1-356. Therefore, the binding sites on DPPIV of ADA and antibodies that inhibit ADA binding are discontinuous and overlapping. Moreover, the 47 and 97 residue spacing of amino acids in these binding sites concords with their location on a beta propeller fold consisting of repeated beta sheets of about 50 amino acids.