Main structural and functional features of the basic cytosolic bovine 21 kDa protein delineated through Hydrophobic Cluster Analysis and molecular modelling

Abstract

A 21 kDa protein purified from bovine brain cytosol was previously described as a hydrophobic ligand binding protein; however, its accurate biological function remained still uncertain. In order to get further information about its potential biological role, an extended prediction of its secondary and three dimensional structures was undertaken. We describe here a process which permitted us to discover a structural homology between the 21 kDa protein and the N-domain of yeast phosphoglycerate kinase (PGK). This process is based on comparing the 21 kDa protein with all the proteins presenting a slight homology, by using the Hydrophobic Cluster Analysis (HCA) method. According to the observed similarity between the N-domain of yeast PGK and the 21 kDa protein, we built a model which was shown to possess a potential binding site for nucleotides. Moreover, the model obtained presents three-dimensional (3D) structure similarity with adenylate kinase. These results suggest two main hypotheses: (i) the 21 kDa protein may belong to the kinase family; (ii) the binding of a nucleotide could imply a modification of the 3D structure of the 21 kDa protein that can promote the transfer of hydrophobic ligands to the plasma membrane. Meanwhile, verification of these hypotheses has been in part performed experimentally: the 21 kDa protein binds MgATP as well as, to a lesser extent, phosphoglycerate.

Neuronal expression of hippocampal cholinergic neurostimulating peptide (HCNP)-precursor mRNA in rat brain

“…We recently reported that the amino-acid sequences of rat and human HCNP-precursor proteins, deduced from the respective cDNAs , were highly homologous with those of the bovine phosphatidylethanolamine-binding protein (Schoentgen et al, 1987) and the rat brain 23-kDa protein associated with opioid pep-consensus sequence for an ATP binding site (Schoentgen et al, 1992;Tohdoh et al, 1995), and we have shown that the precursor proteins of murine and human origin do indeed bind ATP .…”

“…, 1987), and is indistinguishable from the partial sequence of a 23-kDa plasma membrane protein from rat sperm (Jones and Hall, 1991), as well as from a rat 23-kDa protein associated with an opioid peptide (Grandy et al, 1990). Moreover, it has been suggested that the phosphatidylethanolamine-binding protein has a consensus sequence of an ATP-binding site (Schoentgen et al, 1992), and we found that the HCNPprecursor proteins of rats and humans also contain such a motif, and that both have a specific affinity for ATP, but not for any other nucleotide . In more recent studies in which a radioimmunoassay (RIA) and a combination of RIA and high-pressure liquid chromatography were used, we demonstrated the presence of HCNP and its precursor protein in a variety of rat tissues, with amounts in neuronal tissues examined inversely related to age of the animal .…”

Amino acid sequence of the Homo sapiens brain 21-23-kDa protein (Neuropolypeptide h3), comparison with its counterparts from Rattus norvegicus and Bos taurus species, and expression of its mRNA in different tissues

“…Thus, the building of a 21-kDa model was carried out from the crystallographic data of the Saccharomyces cerevisiae phosphoglycerate kinase. The model revealed a potential nucleotide binding site which was confirmed by biochemical tests, suggesting that the 21-kDa protein may be involved in phosphorylation or modulation mechanisms (Schoentgen et al 1992). More recently, Grandy et al (1990) isolated a 23-kDa protein from R. norvegicus brain membranes and established the sequence of a cDNA encoding this protein.…”

From structure to function: possible biological roles of a new widespread protein family binding hydrophobic ligands and displaying a nucleotide binding site

“…Furthermore, when comparing the amino acid sequences of the 21 23 kDa protein family members, the central region constituted by residues 60 126 appeared to be particularly well conserved (see Fig. 1); interestingly, this central region corresponds to the potential nucleotide binding site observed in the bovine protein model [18]. The nucleotide site may be implicated in the binding of the bovine brain 21 23 kDa protein to small GTP-binding proteins [19]; the possibility for the mammalian protein to bind small G proteins is in accordance with the observation that TFS1 seems to be implicated in pathway modulations involving small GTPbinding proteins.…”

“…Based on this result, a molecular model of the bovine 21-23 kDa protein was built from the crystallographic data of the Saccharomyces cerevisiae phosphoglycerate kinase. This model contained a consensus sequence and three-dimensional folding corresponding to a potential nucleotide binding site [18]. The binding of nucleotides (especially FMN and GTP) to the bovine brain 21-23 kDa protein was then demonstrated by affinity chromatography [19].…”