Crystal structures of two Sm protein complexes and their implications for the assembly of the spliceosomal snRNPs. (9/22435)

The U1, U2, U4/U6, and U5 small nuclear ribonucleoprotein particles (snRNPs) involved in pre-mRNA splicing contain seven Sm proteins (B/B', D1, D2, D3, E, F, and G) in common, which assemble around the Sm site present in four of the major spliceosomal small nuclear RNAs (snRNAs). These proteins share a common sequence motif in two segments, Sm1 and Sm2, separated by a short variable linker. Crystal structures of two Sm protein complexes, D3B and D1D2, show that these proteins have a common fold containing an N-terminal helix followed by a strongly bent five-stranded antiparallel beta sheet, and the D1D2 and D3B dimers superpose closely in their core regions, including the dimer interfaces. The crystal structures suggest that the seven Sm proteins could form a closed ring and the snRNAs may be bound in the positively charged central hole.  (+info)

Crystal structure of the cytoplasmic domain of the type I TGF beta receptor in complex with FKBP12. (10/22435)

Activation of the type I TGFbeta receptor (TbetaR-I) requires phosphorylation of a regulatory segment known as the GS region, located upstream of the serine/threonine kinase domain in the cytoplasmic portion of the receptor. The crystal structure of a fragment of unphosphorylated TbetaR-I, containing both the GS region and the catalytic domain, has been determined in complex with the FK506-binding protein FKBP12. TbetaR-I adopts an inactive conformation that is maintained by the unphosphorylated GS region. FKBP12 binds to the GS region of the receptor, capping the TbetaR-II phosphorylation sites and further stabilizing the inactive conformation of TbetaR-I. Certain structural features at the catalytic center of TbetaR-I are characteristic of tyrosine kinases rather than Ser/Thr kinases.  (+info)

Phe161 and Arg166 variants of p-hydroxybenzoate hydroxylase. Implications for NADPH recognition and structural stability. (11/22435)

Phe161 and Arg166 of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens belong to a newly discovered sequence motif in flavoprotein hydroxylases with a putative dual function in FAD and NADPH binding [1]. To study their role in more detail, Phe161 and Arg166 were selectively changed by site-directed mutagenesis. F161A and F161G are catalytically competent enzymes having a rather poor affinity for NADPH. The catalytic properties of R166K are similar to those of the native enzyme. R166S and R166E show impaired NADPH binding and R166E has lost the ability to bind FAD. The crystal structure of substrate complexed F161A at 2.2 A is indistinguishable from the native enzyme, except for small changes at the site of mutation. The crystal structure of substrate complexed R166S at 2.0 A revealed that Arg166 is important for providing an intimate contact between the FAD binding domain and a long excursion of the substrate binding domain. It is proposed that this interaction is essential for structural stability and for the recognition of the pyrophosphate moiety of NADPH.  (+info)

Dengue virus NS3 serine protease. Crystal structure and insights into interaction of the active site with substrates by molecular modeling and structural analysis of mutational effects. (12/22435)

The mosquito-borne dengue viruses are widespread human pathogens causing dengue fever, dengue hemorrhagic fever, and dengue shock syndrome, placing 40% of the world's population at risk with no effective treatment. The viral genome is a positive strand RNA that encodes a single polyprotein precursor. Processing of the polyprotein precursor into mature proteins is carried out by the host signal peptidase and by NS3 serine protease, which requires NS2B as a cofactor. We report here the crystal structure of the NS3 serine protease domain at 2.1 A resolution. This structure of the protease combined with modeling of peptide substrates into the active site suggests identities of residues involved in substrate recognition as well as providing a structural basis for several mutational effects on enzyme activity. This structure will be useful for development of specific inhibitors as therapeutics against dengue and other flaviviral proteases.  (+info)

Crystal structure of wild-type human procathepsin K. (13/22435)

Cathepsin K is a lysosomal cysteine protease belonging to the papain superfamily. It has been implicated as a major mediator of osteoclastic bone resorption. Wild-type human procathepsin K has been crystallized in a glycosylated and a deglycosylated form. The latter crystals diffract better, to 3.2 A resolution, and contain four molecules in the asymmetric unit. The structure was solved by molecular replacement and refined to an R-factor of 0.194. The N-terminal fragment of the proregion forms a globular domain while the C-terminal segment is extended and shows substantial flexibility. The proregion interacts with the enzyme along the substrate binding groove and along the proregion binding loop (residues Ser138-Asn156). It binds to the active site in the opposite direction to that of natural substrates. The overall binding mode of the proregion to cathepsin K is similar to that observed in cathepsin L, caricain, and cathepsin B, but there are local differences that likely contribute to the specificity of these proregions for their cognate enzymes. The main observed difference is in the position of the short helix alpha3p (67p-75p), which occupies the S' subsites. As in the other proenzymes, the proregion utilizes the S2 subsite for anchoring by placing a leucine side chain there, according to the specificity of cathepsin K toward its substrate.  (+info)

Crystal structure of human muscle aldolase complexed with fructose 1,6-bisphosphate: mechanistic implications. (14/22435)

Fructose 1,6-bisphosphate aldolase catalyzes the reversible cleavage of fructose 1,6-bisphosphate and fructose 1-phosphate to dihydroxyacetone phosphate and either glyceraldehyde 3-phosphate or glyceraldehyde, respectively. Catalysis involves the formation of a Schiff's base intermediate formed at the epsilon-amino group of Lys229. The existing apo-enzyme structure was refined using the crystallographic free-R-factor and maximum likelihood methods that have been shown to give improved structural results that are less subject to model bias. Crystals were also soaked with the natural substrate (fructose 1,6-bisphosphate), and the crystal structure of this complex has been determined to 2.8 A. The apo structure differs from the previous Brookhaven-deposited structure (1ald) in the flexible C-terminal region. This is also the region where the native and complex structures exhibit differences. The conformational changes between native and complex structure are not large, but the observed complex does not involve the full formation of the Schiff's base intermediate, and suggests a preliminary hydrogen-bonded Michaelis complex before the formation of the covalent complex.  (+info)

Crystal structure of the FMN-binding domain of human cytochrome P450 reductase at 1.93 A resolution. (15/22435)

The crystal structure of the FMN-binding domain of human NADPH-cytochrome P450 reductase (P450R-FMN), a key component in the cytochrome P450 monooxygenase system, has been determined to 1.93 A resolution and shown to be very similar both to the global fold in solution (Barsukov I et al., 1997, J Biomol NMR 10:63-75) and to the corresponding domain in the 2.6 A crystal structure of intact rat P450R (Wang M et al., 1997, Proc Nat Acad Sci USA 94:8411-8416). The crystal structure of P450R-FMN reported here confirms the overall similarity of its alpha-beta-alpha architecture to that of the bacterial flavodoxins, but reveals differences in the position, number, and length of the helices relative to the central beta-sheet. The marked similarity between P450R-FMN and flavodoxins in the interactions between the FMN and the protein, indicate a striking evolutionary conservation of the FMN binding site. The P450R-FMN molecule has an unusual surface charge distribution, leading to a very strong dipole, which may be involved in docking cytochrome P450 into place for electron transfer near the FMN. Several acidic residues near the FMN are identified by mutagenesis experiments to be important for electron transfer to P4502D6 and to cytochrome c, a clear indication of the part of the molecular surface that is likely to be involved in substrate binding. Somewhat different parts are found to be involved in binding cytochrome P450 and cytochrome c.  (+info)

Comparison of the backbone dynamics of the apo- and holo-carboxy-terminal domain of the biotin carboxyl carrier subunit of Escherichia coli acetyl-CoA carboxylase. (16/22435)

The biotin carboxyl carrier protein (BCCP) is a subunit of acetyl-CoA carboxylase, a biotin-dependent enzyme that catalyzes the first committed step of fatty acid biosynthesis. In its functional cycle, this protein engages in heterologous protein-protein interactions with three distinct partners, depending on its state of post-translational modification. Apo-BCCP interacts specifically with the biotin holoenzyme synthetase, BirA, which results in the post-translational attachment of biotin to a single lysine residue on BCCP. Holo-BCCP then interacts with the biotin carboxylase subunit of acetyl-CoA carboxylase, which leads to the addition of the carboxylate group of bicarbonate to biotin. Finally, the carboxy-biotinylated form of BCCP interacts with transcarboxylase in the transfer of the carboxylate to acetyl-CoA to form malonyl-CoA. The determinants of protein-protein interaction specificity in this system are unknown. The NMR solution structure of the unbiotinylated form of an 87 residue C-terminal domain fragment (residue 70-156) of BCCP (holoBCCP87) and the crystal structure of the biotinylated form of a C-terminal fragment (residue 77-156) of BCCP from Escherichia coli acetyl-CoA carboxylase have previously been determined. Comparative analysis of these structures provided evidence for small, localized conformational changes in the biotin-binding region upon biotinylation of the protein. These structural changes may be important for regulating specific protein-protein interactions. Since the dynamic properties of proteins are correlated with local structural environments, we have determined the relaxation parameters of the backbone 15N nuclear spins of holoBCCP87, and compared these with the data obtained for the apo protein. The results indicate that upon biotinylation, the inherent mobility of the biotin-binding region and the protruding thumb, with which the biotin group interacts in the holo protein, are significantly reduced.  (+info)