The principal heat-shock proteins that have chaperone activity (that is, they protect newly made proteins from misfolding) belong to five conserved classes: HSP100, HSP90, HSP70, HSP60 and the small heat-shock proteins (sHSPs). The sHSPs can form large multimeric structures and have a wide range of cellular functions, including endowing cells with thermotolerance in vivo and being able to act as molecular chaperones in vitro; sHSPs do this by forming stable complexes with folding intermediates of their protein substrates. However, there is little information available about these structures or the mechanism by which substrates are protected from thermal denaturation by sHSPs. Here we report the crystal structure of a small heat-shock protein from Methanococcus jannaschii, a hyperthermophilic archaeon. The monomeric folding unit is a composite beta-sandwich in which one of the beta-strands comes from a neighbouring molecule. Twenty-four monomers form a hollow spherical complex of octahedral symmetry, with eight trigonal and six square 'windows'. The sphere has an outer diameter of 120 A and an inner diameter of 65 A.

The activity of an oncoprotein and the secretion of a pheromone can be affected by an unusual protein modification. Specifically, posttranslational modification of yeast a-factor and Ras protein requires an intermediate of the cholesterol biosynthetic pathway. This modification is apparently essential for biological activity. Studies of yeast mutants blocked in sterol biosynthesis demonstrated that the membrane association and biological activation of the yeast Ras2 protein require mevalonate, a precursor of sterols and other isoprenes such as farnesyl pyrophosphate. Furthermore, drugs that inhibit mevalonate biosynthesis blocked the in vivo action of oncogenic derivatives of human Ras protein in the Xenopus oocyte assay. The same drugs and mutations also prevented the posttranslational processing and secretion of yeast a-factor, a peptide that is farnesylated. Thus, the mevalonate requirement for Ras activation may indicate that attachment of a mevalonate-derived (isoprenoid) moiety to Ras proteins is necessary for membrane association and biological function. These observations establish a connection between the cholesterol biosynthetic pathway and transformation by the ras oncogene and offer a novel pharmacological approach to investigating, and possibly controlling, ras-mediated malignant transformations.

Many small bacterial, archaebacterial, and eukaryotic genomes have been sequenced, and the larger eukaryotic genomes are predicted to be completely sequenced within the next decade. In all genomes sequenced to date, a large portion of these organisms' predicted protein coding regions encode polypeptides of unknown biochemical, biophysical, and͞or cellular functions. Three-dimensional structures of these proteins may suggest biochemical or biophysical functions. Here we report the crystal structure of one such protein, MJ0577, from a hyperthermophile, Methanococcus jannaschii, at 1.7-Å resolution. The structure contains a bound ATP, suggesting MJ0577 is an ATPase or an ATP-mediated molecular switch, which we confirm by biochemical experiments. Furthermore, the structure reveals different ATP binding motifs that are shared among many homologous hypothetical proteins in this family. This result indicates that structure-based assignment of molecular function is a viable approach for the large-scale biochemical assignment of proteins and for discovering new motifs, a basic premise of structural genomics.As of October 1998, 16 microbial genomes had been completely sequenced (Web site: www.tigr.org). These genomes are from all three branches of life: four from the Archaea, one from Eukarya, and the rest from Bacteria. To predict a function for each of their predicted protein coding regions or ORFs, the amino acid sequence of the ORF is compared against all functionally assigned sequences in protein sequence databases. If there is significant sequence or motif identity between the ORF and a functionally assigned sequence, then it is assumed that the two sequences share the same function. Unfortunately, up to 62% of the ORFs from these genomes share little or no sequence identity with any assigned sequence and hence are of unknown function (1-15). A major challenge, therefore, is to find ways to reliably and rapidly predict or determine the molecular (biochemical and biophysical) functions as well as cellular functions of these proteins.One approach for assigning the molecular function of a protein with unknown function is first to determine the three-dimensional structure of the protein by either x-ray crystallography or NMR. The structure, instead of the amino acid sequence, then is compared against those of the protein structure database (Protein Data Bank). If there are one or more significant structural homologs, the hypothetical protein is predicted to have molecular properties similar to the homologs. The predictions then can be tested experimentally. The molecular function then can provide a basis for searching for the cellular function of the protein. This method, structural genomics (16,17), is far more sensitive than primary sequence comparisons because proteins having insignificant sequence similarity often adopt similar tertiary structures with similar or related molecular functions. With the increasing advances in computer hardware and software associated with structure determination, this approach ...