Topology of acyltransferase motifs and substrate specificity and accessibility in 1-acyl-sn-glycero-3-phosphate acyltransferase 1

1-acyl-sn-glycero-3-phosphate (AGP) acyltransferases (AGPAT) are involved in de novo biosynthesis of glycerolipids, such as phospholipids and triacylglycerol. Alignment of amino acid sequences from AGPAT, sn-glycerol-3-phosphate acyltransferase, and dihydroxyacetonephosphate acyltransferase reveals four regions with strong homology (acyltransferase motifs I-IV). The invariant amino acids within these regions may be part of a catalytically important site in this group of acyl-CoA acyltransferases. However, in human AGPAT1 a transmembrane domain is predicted to separate motif I on the cytosolic side from motifs II-III on the lumenal side, with motif IV near surface of the membrane. The topology of motifs I and III was confirmed by experiments with recombinant AGPAT1 containing potential glycosylation site near the motifs. This topology conflicts with the expectation that catalytically important sites are near one another, raising questions of whether the acyltransferase motifs really are important for AGPAT catalysis, and how substrates access motifs II-III on the lumenal side of the endoplasmic reticulum membrane. Using human AGPAT1 as a model, we have examined the catalytic roles of highly conserved residues in the four acyltransferase motifs by site-directed mutagenesis. Modifications of the sidechain structures of His104, Asp109, Phe146, Arg149, Glu178, Gly179, Thr180, Arg181 and Ile208 all affected AGPAT1 activity, indicating that the acyltransferase motifs indeed are important for AGPAT catalysis. In addition, we examined substrate accessibility to the catalytic domain of human AGPAT1 using a competition assay. Lysophosphatidic acid (LPA) with fatty acid chains shorter than 10 carbons did not access the catalytic domain, suggesting that LPA hydrophobicity is important. In contrast, short chain acyl-CoAs did access the catalytic domain but did not serve as the second substrate. These results suggest that motifs II and III are involved in LPA binding and motifs I and IV are involved in acyl-CoA binding.     

The crystal and molecular structure of human annexin V, an anticoagulant protein that binds to calcium and membranes

Human annexin V (PP4), a member of the family of calcium, membrane binding proteins, has been crystallized in the presence of calcium and analysed by crystallography by multiple isomorphic replacement at 3 A and preliminarily refined at 2.5 A resolution. The molecule has dimensions of 64 x 40 x 30 A3 and is folded into four domains of similar structure. Each domain consists of five alpha-helices wound into a right-handed superhelix yielding a globular structure of approximately 18 A diameter. The domains have hydrophobic cores whose amino acid sequences are conserved between the domains and within the annexin family of proteins. The four domains are folded into an almost planar array by tight (hydrophobic) pair-wise packing of domains II and III and I and IV to generate modules (II-III) and (I-IV), respectively. The assembly is symmetric with three parallel approximate diads relating II to III, I to IV and the module (II-III) to (I-IV), respectively. The latter diad marks a channel through the centre of the molecule coated with charged amino acid residues. The protein has structural features of channel forming membrane proteins and a polar surface characteristic of soluble proteins. It is a member of the third class of amphipathic proteins different from soluble and membrane proteins.     

Analysis of the receptor-binding site of murine coronavirus spike protein.

It has been found that a domain composed of 330 amino acids of the N terminus of murine coronavirus spike protein [S1N(330)] is involved in receptor-binding activity (H. Kubo, Y.K. Yamada, and F. Taguchi, J. Virol. 68:5403-5410, 1994). To delineate the amino acid sequences involved in receptor-binding activity, we have compared the S1N(330) proteins of seven different mouse hepatitis virus MHV strains that are able to utilize the MHV receptor protein. Three conserved regions (sites I, II, and III) were found to consist of more than 10 identical amino acids, and they were analyzed for receptor-binding activity by site-directed mutagenesis. S1N(330) with a substitution at position 62 from the N terminus of S1 in region I and that with substitutions at positions 212, 214, and 216 in region II showed no receptor-binding activity. The S1N(330) mutants without receptor-binding activity were not able to prevent virus binding to the receptor. These results suggest that the receptor-binding site on S1N(330) is composed of regions located apart from each other in the protein's primary structure, in which Thr at position 62 as well as amino acids located at positions 212, 214, and 216 are particularly important.     

A chimaeric tryptophan decarboxylase gene as a novel selectable marker in plant cells

A cDNA clone, pMA1949, detects two mRNA species in wheat seedling tissue that are late embryogenesis-abundant (LEA) and dehydration stress-inducible. Sequence analysis of the pMA1949 clone shows it to be a 991 bp partial cDNA encoding a polypeptide of 317 amino acids with homology to two group 3 LEA proteins, carrot (DC8) and a soybean protein encoded by pGmPM2 cDNA. Molecular analysis of the deduced protein reveals a 33 kDa acidic and extremely hydrophilic protein with potential amphiphilic -helical regions. In addition, the protein contains eleven similar, contiguous repeats of 11 amino acids, which are separated by 118 amino acids from two additional and unique repeats of 36 residues each at the carboxyl end of the protein. Comparisons of sequences of reported group 3 LEA proteins revealed that there are two types, separable by sequence similarity of the 11 amino acid repeating motifs and by the presence or absence of a certain amino acid stretch at the carboxyl terminus. Based on resuls from these comparisons, we propose a second type of group 3 LEA proteins, called group 3 LEA (II).