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Success of mitosis depends upon the coordinated and regulated activity of many cellular factors, including kinesin motor proteins, which are required for the assembly and function of the mitotic spindle. Eg5 is a kinesin implicated in the formation of the bipolar spindle and its movement prior to and during anaphase. We have determined the crystal structure of the Eg5 motor domain with ADP-Mg bound. This structure revealed a new intramolecular binding site of the neck-linker. In other kinesins, the neck-linker has been shown to be a critical mechanical element for force generation. The neck-linker of conventional kinesin is believed to undergo an ordered-to-disordered transition as it translocates along a microtubule. The structure of Eg5 showed an ordered neck-linker conformation in a position never observed previously. The docking of the neck-linker relies upon residues conserved only in the Eg5 subfamily of kinesin motors. Based on this new information, we suggest that the neck-linker of Eg5 may undergo an ordered-to-ordered transition during force production. This ratchet-like mechanism is consistent with the biological activity of Eg5.

Previous mutagenesis studies along with molecular modeling using the x-ray coordinates of the rabbit 15-lipoxygenase have led to the suggestion that the size of the substrate binding pocket may play an essential role in determining the oxygenation specificity of 5-, 12-, and 15-lipoxygenases. Based on the x-ray crystal structure of rabbit 15-lipoxygenase, Ile(593) appeared to be important in defining size and shape of the substrate-binding site in 15-lipoxygenases. We found that substitution of Ile(593) with alanine shifted the positional specificity of this enzyme toward 12-lipoxygenation. To compare the importance of position 593 with previously defined determinants for the oxygenation specificity, we introduced small (alanine-scan) or large amino acids (phenylalanine-scan) at critical positions surrounding the putative fatty acid-binding site, so that the volume of the pocket was either increased or decreased. Enlargement or alteration in packing density within the substrate binding pocket in the rabbit 15-lipoxygenase increased the share of 12-lipoxygenase products, whereas a smaller active site favored 15-lipoxygenation. Simultaneous substitution of both large and small residues in the context of either a 15- or 12-lipoxygenase indicated that there is a functional interplay of the sequence determinants for lipoxygenation specificity. If the 15-lipoxygenase active site is enlarged excessively, however, no lipoxygenation was observed anymore. Together these results indicate the importance of the overall size and shape of the arachidonic acid binding pocket in defining the specificity of lipoxygenase reaction.

Ncd is a microtubule minus-end directed motor of the kinesin superfamily. Previously it has been shown that ncd and kinesin motor domains share the same major binding site on microtubules. Here we report a three-dimensional EM reconstruction of negatively stained two-dimensional Zn-induced tubulin crystal sheets (Zn-sheets) decorated with the ncd motor domain at a resolution of 16 A˚. This work has revealed a second specific binding site for the ncd motor domain. The motor binding site on the tubulin Zn-sheets spans both α and β tubulin subunits. This binding site is located at a position different from the previously identified ncd binding site on microtubules and may play a role in motor function.

A comparison of the refined crystal structures of dimeric glycogen phosphorylase b and a reveals structural changes that represent the first step in the activation of the enzyme. On phosphorylation of serine-14, the N-terminus of each subunit assumes an ordered helical conformation and binds to the surface of the dimer. The consequent structural changes at the N- and C-terminal regions lead to strengthened interactions between subunits and alter the binding sites for allosteric effectors and substrates.

Combinatorial regulation of transcription implies flexible yet precise assembly of multiprotein regulatory complexes in response to signals. Biochemical and crystallographic analyses revealed that hormone binding leads to the formation of a hydrophobic groove within the ligand binding domain (LBD) of the thyroid hormone receptor that interacts with an LxxLL motif-containing alpha-helix from GRIP1, a coactivator. Residues immediately adjacent to the motif modulate the affinity of the interaction; the motif and the adjacent sequences are employed to different extents in binding to different receptors. Such interactions of amphipathic alpha-helices with hydrophobic grooves define protein interfaces in other regulatory complexes as well. We suggest that these common structural elements impart flexibility to combinatorial regulation, whereas side chains at the interface impart specificity.

We have cloned the cDNA encoding glycogen phosphorylase (1,4-alpha-D-glucan:orthophosphate alpha-D-glucosyl-transferase, EC 2.4.1.1) from human liver. Blot-hybridization analysis using a large fragment of the cDNA to probe mRNA from rabbit brain, muscle, and liver tissues shows preferential hybridization to liver RNA. Determination of the entire nucleotide sequence of the liver message has allowed a comparison with the previously determined rabbit muscle phosphorylase sequence. Despite an amino acid identity of 80%, the two cDNAs exhibit a remarkable divergence in G+C content. In the muscle phosphorylase sequence, 86% of the nucleotides at the third codon position are either deoxyguanosine or deoxycytidine residues, while in the liver homolog the figure is only 60%, resulting in a strikingly different pattern of codon usage throughout most of the sequence. The liver phosphorylase cDNA appears to represent an evolutionary mosaic; the segment encoding the N-terminal 80 amino acids contains greater than 90% G+C at the third codon position. A survey of other published mammalian cDNA sequences reveals that the data for liver and muscle phosphorylases reflects a bias in codon usage patterns in liver and muscle coding sequences in general.

The cDNA for human muscle glycogen synthase encodes a protein of 737 amino acids. The primary structure of glycogen synthase is not related either to bacterial glycogen synthase or to any glycogen phosphorylase. All nine of the serines that are phosphorylated in the rabbit muscle enzyme in vivo are conserved in the human muscle sequence. The amino- and carboxyl-terminal fragments, which contain all the phosphorylation sites, are very negatively charged. Overall the unphosphorylated protein has a charge of -13, while the fully phosphorylated inactive protein has a net charge of -31. The importance of the asymmetrical charge distribution is discussed.

From a 2.7-A resolution electron density map we have built a model of the polypeptide backbone of a monomer of yeast hexokinase B (EC 2.7.1.1). This map was obtained from a third crystal form of hexokinase, called BIII, which exhibits space group P212121 and which contains only one monomer per asymmetric unit. The 51,000 molecular weight monomer has an elongated shape (80 A by 55 A by 50 A) and is divided into two lobes by a deep central cleft. The polypeptide chain is folded into three structural domains, one of which is predominantly alpha-helical and two of which each contain a beta-pleated sheet flanked by alpha-helices. Both glucose and AMP bind to these crystals and produce significant alterations in the protein structure. Glucose binds in the deep cleft, as was observed previously in the BII crystal of the dimeric enzyme. AMP, however, binds to a site that is different from the major intersubunit ATP binding site observed in the crystalline dimer. The AMP is found near one of the beta-pleated sheets. From our current interpretation of this electron density map we conclude that neither of the two nucleotide binding regions has the same structure as has been observed for the nucleotide binding regions of the dehydrogenases, adenylate kinase, and phosphoglycerate kinase, although some similarities exist.

We have cloned the cDNA encoding a new isozyme of glycogen phosphorylase (1,4-D-glucan:orthosphosphate D-glucosyltransferase, EC 2.4.1.1) from a cDNA library prepared from a human brain astrocytoma cell line. Blot-hybridization analysis reveals that this message is preferentially expressed in human brain, but is also found at a low level in human fetal liver and adult liver and muscle tissues. Although previous studies have suggested that the major isozyme of phosphorylase found in all fetal tissues is the brain type, our data show that the predominant mRNA in fetal liver (24-week gestation) is the adult liver form. The protein sequence deduced from the nucleotide sequence of the brain phosphorylase cDNA is 862 amino acids long compared with 846 and 841 amino acids for the liver and muscle isozymes, respectively; the greater length of brain phosphorylase is entirely due to an extension at the far C-terminal portion of the protein. The muscle and brain isozymes share greater identity with regard to nucleotide and deduced amino acid sequences, codon usage, and nucleotide composition than either do with the liver sequence, suggesting a closer evolutionary relationship between them. Spot blot hybridization of the brain phosphorylase cDNA to laser-sorted human chromosome fractions, and Southern blot analysis of hamster/human hybrid cell line DNA reveals that the exact homolog of the newly cloned cDNA maps to chromosome 20, but that a slightly less homologous gene is found on chromosome 10 as well. The liver and muscle genes have previously been localized to chromosomes 14 and 11, respectively. This suggests that the phosphorylase genes evolved by duplication and translocation of a common ancestral gene, leading to divergence of elements controlling gene expression and of structural features of the phosphorylase proteins that confer tissue-specific functional properties.

The bifunctional rat liver enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (ATP:D-fructose-6-phosphate 2-phosphotransferase/D-fructose-2,6-bisphosphate 2-phosphohydrolase, EC 2.7.1.105/EC 3.1.3.46) is constructed of two independent catalytic domains. We present evidence that the kinase and bisphosphatase halves of the bifunctional enzyme are, respectively, structurally similar to the glycolytic enzymes 6-phosphofructo-1-kinase and phosphoglycerate mutase. Computer-assisted modeling of the C-terminal bisphosphatase domain reveals a hydrophobic core and active site residue constellation equivalent to the yeast mutase structure; structural differences map to length-variable, surface-located loops. Sequence patterns derived from the structural alignment of mutases and the bisphosphatase further detect a significant similarity to a family of acid phosphatases. The N-terminal kinase domain, in turn, is predicted to form a nucleotide-binding fold that is analogous to a segment of 6-phosphofructo-1-kinase, suggesting that these unrelated enzymes bind fructose 6-phosphate and ATP substrates in a similar geometry. This analysis indicates that the bifunctional enzyme is the likely product of gene fusion of kinase and mutase/phosphatase catalytic units.

Sterol carrier protein 2 (SCP2) is involved in the later steps of cholesterol biosynthesis and in the intracellular transport of cholesterol. In the present investigation, the amino acid sequence of SCP2 from rat liver has been determined. It is a single polypeptide chain with 122 amino acid residues. Secondary structure prediction indicates an amphipathic alpha-helix region for residues 21-34 and antiparallel beta-sheet structure for residues 35-95. A major finding is the significant homology which exists over approximately 80 residues between SCP2 and the variable domains of the heavy chain of immunoglobulin G.

Proteases that are encoded by animal picornaviruses and plant como- and potyviruses form a related group of cysteine-active-center enzymes that are essential for virus maturation. We show that these proteins are homologous to the family of trypsin-like serine proteases. In our model, the active-site nucleophile of the trypsin catalytic triad, Ser-195, is changed to a Cys residue in these viral proteases. The other two residues of the triad, His-57 and Asp-102, are otherwise absolutely conserved in all the viral protease sequences. Secondary structure analysis of aligned sequences suggests the location of the component strands of the twin beta-barrel trypsin fold in the viral proteases. Unexpectedly, the 2a and 3c subclasses of viral cysteine proteases are, respectively, homologous to the small and large structural subclasses of trypsin-like serine proteases. This classification allows the molecular mapping of residues from viral sequences onto related tertiary structures; we precisely identify amino acids that are strong determinants of specificity for both small and large viral cysteine proteases.