Biomedical subjects

Florante A Quiocho

Publications and source records attributed to Florante A Quiocho.

At least 19 recordsLinked to original sources

PubMed2006

Crystal structures of the vaccinia virus polyadenylate polymerase heterodimer: insights into ATP selectivity and processivity.

Polyadenylation of mRNAs in poxviruses, crucial for virion maturation, is carried out by a poly(A) polymerase heterodimer composed of a catalytic component, VP55, and a processivity factor, VP39. The ATP-gamma-S bound and unbound crystal structures of the vaccinia polymerase reveal an unusual architecture for VP55 that comprises of N-terminal, central or catalytic, and C-terminal domains with different topologies and that differs from many polymerases, including the eukaryotic poly(A) polymerases. Residues in the active site of VP55, located between the catalytic and C-terminal domains, make specific interactions with the adenine of the ATP analog, establishing the molecular basis of ATP recognition. VP55's concave surface docks the globular VP39. A model for RNA primer binding that involves all three VP55 domains and VP39 is proposed. The model supports biochemical evidence that VP39 functions as a processivity factor by partially enclosing the RNA primer at the heterodimer interface.

Adenosine Triphosphate↗

PubMed2006

Multipurpose MRG domain involved in cell senescence and proliferation exhibits structural homology to a DNA-interacting domain.

The ubiquitous MRG/MORF family of proteins is involved in cell senescence, or the terminal loss of proliferative potential, a model for aging and tumor suppression at the cellular level. These proteins are defined by the approximately 20 kDa MRG domain that binds a plethora of transcriptional regulators and chromatin-remodeling factors, including the histone deacetylase transcriptional corepressor mSin3A and the novel nuclear protein PAM14, and they are also known components of the Tip60/NuA4 complex via interactions with the MRG binding protein (MRGBP). We present here the crystal structure of a prototypic MRG domain from human MRG15 whose core consists of two orthogonal helix hairpins. Despite the lack of sequence similarity, the core structure has surprisingly striking homology to a DNA-interacting domain of the tyrosine site-specific recombinases XerD, lambda integrase, and Cre. Site-directed mutagenesis studies based on the X-ray structure and bioinformatics identified key residues involved in the binding of PAM14 and MRGBP.

Amino Acid Sequence↗

PubMed2006

Structural characterization of the UL25 DNA-packaging protein from herpes simplex virus type 1.

Herpesviruses replicate their double stranded DNA genomes as high-molecular-weight concatemers which are subsequently cleaved into unit-length genomes by a complex mechanism that is tightly coupled to DNA insertion into a preformed capsid structure, the procapsid. The herpes simplex virus type 1 UL25 protein is incorporated into the capsid during DNA packaging, and previous studies of a null mutant have demonstrated that its function is essential at the late stages of the head-filling process, either to allow packaging to proceed to completion or for retention of the viral genome within the capsid. We have expressed and purified an N-terminally truncated form of the 580-residue UL25 protein and have determined the crystallographic structure of the region corresponding to amino acids 134 to 580 at 2.1-Angstroms resolution. This structure, the first for any herpesvirus protein involved in processing and packaging of viral DNA, reveals a novel fold, a distinctive electrostatic distribution, and a unique "flexible" architecture in which numerous flexible loops emanate from a stable core. Evolutionary trace analysis of UL25 and its homologues in other herpesviruses was used to locate potentially important amino acids on the surface of the protein, leading to the identification of four putative docking regions for protein partners.

Amino Acid Sequence↗

PubMed2005

Sec15 interacts with Rab11 via a novel domain and affects Rab11 localization in vivo.

Sec15, a component of the exocyst, recognizes vesicle-associated Rab GTPases, helps target transport vesicles to the budding sites in yeast and is thought to recruit other exocyst proteins. Here we report the characterization of a 35-kDa fragment that comprises most of the C-terminal half of Drosophila melanogaster Sec15. This C-terminal domain was found to bind a subset of Rab GTPases, especially Rab11, in a GTP-dependent manner. We also provide evidence that in fly photoreceptors Sec15 colocalizes with Rab11 and that loss of Sec15 affects rhabdomere morphology. Determination of the 2.5-A crystal structure of the C-terminal domain revealed a novel fold consisting of ten alpha-helices equally distributed between two subdomains (N and C subdomains). We show that the C subdomain, mainly via a single helix, is sufficient for Rab binding.

Animals↗

PubMed2005

Ligand-free and -bound structures of the binding protein (LivJ) of the Escherichia coli ABC leucine/isoleucine/valine transport system: trajectory and dynamics of the interdomain rotation and ligand specificity.

The leucine/isoleucine/valine-binding protein (LIVBP or LivJ) serves as the primary high-affinity receptor of the Escherichia coli ABC-type transporter for the three aliphatic amino acids. The first structure of LIVBP determined previously without bound ligand showed a molecule comprised of two domains which are far apart and bisected by a wide open, solvent-accessible cleft. Here we report the crystal structures of another ligand-free state and three complexes with the aliphatic amino acids. In the present ligand-free structure, the two domains are farther apart. In the three very similar complex structures, the two domains are in close proximity, and each desolvated ligand is completely engulfed in the cleft and bound by both domains. The two different ligand-free structures, combined with those of the very similar ligand-bound structures, indicate the trajectory and backbone torsion angle changes of the hinges that accompany domain closure and play crucial functional roles. The amino acids are bound by polar and nonpolar interactions, occurring predominantly in one domain. Consistent with the protein specificity, the aliphatic side chains of the ligands lie in a hydrophobic pocket fully formed following domain or cleft closure. Comparison of the structures of LIVBP with several different binding proteins indicates no correlations between the magnitudes of the hinge-bending angles and the protein masses, the ligand sizes, or the number of segments connecting the two domains. Results of normal-mode analysis and molecular dynamics simulations are consistent with the trajectory and intrinsic flexibility of the interdomain hinges and the dominance of one domain in ligand binding in the open state.

ATP-Binding Cassette Transporters↗

PubMed2005

Structure of calmodulin bound to the hydrophobic IQ domain of the cardiac Ca(v)1.2 calcium channel.

Ca2+-dependent inactivation (CDI) and facilitation (CDF) of the Ca(v)1.2 Ca2+ channel require calmodulin binding to a putative IQ motif in the carboxy-terminal tail of the pore-forming subunit. We present the 1.45 A crystal structure of Ca2+-calmodulin bound to a 21 residue peptide corresponding to the IQ domain of Ca(v)1.2. This structure shows that parallel binding of calmodulin to the IQ domain is governed by hydrophobic interactions. Mutations of residues I1672 and Q1673 in the peptide to alanines, which abolish CDI but not CDF in the channel, do not greatly alter the structure. Both lobes of Ca2+-saturated CaM bind to the IQ peptide but isoleucine 1672, thought to form an intramolecular interaction that drives CDI, is buried. These findings suggest that this structure could represent the conformation that calmodulin assumes in CDF.

Animals↗

PubMed2004

Computational and biochemical identification of a nuclear pore complex binding site on the nuclear transport carrier NTF2.

Nuclear transport carriers interact with proteins of the nuclear pore complex (NPC) to transport their cargo across the nuclear envelope. One such carrier is nuclear transport factor 2 (NTF2), whose import cargo is the small GTPase Ran. A domain highly homologous to the small NTF2 protein (14kDa) is also found in a number of additional proteins, which together make up the NTF2 domain containing superfamily of proteins. Using structural, computational and biochemical analysis we have identified a functional site that is present throughout this superfamily, and our results indicate that this site functions as an NPC binding site in NTF2. Previously we showed that a D23A mutant of NTF2 exhibits increased affinity for the NPC. The mechanism of this mutation, however, was unknown as this region of NTF2 had not been implicated in binding to NPC proteins. Here we show that the D23A mutation in NTF2 does not result in gross structural changes affecting other known NPC binding sites. Instead, the D23 residue is located in an evolutionarily important region in the NTF2 domain containing superfamily, that in NTF2, is involved in binding to the NPC.

Active Transport, Cell Nucleus↗

PubMed2004

Human fatty acid synthase: structure and substrate selectivity of the thioesterase domain.

Human fatty acid synthase is a large homodimeric multifunctional enzyme that synthesizes palmitic acid. The unique carboxyl terminal thioesterase domain of fatty acid synthase hydrolyzes the growing fatty acid chain and plays a critical role in regulating the chain length of fatty acid released. Also, the up-regulation of human fatty acid synthase in a variety of cancer makes the thioesterase a candidate target for therapeutic treatment. The 2.6-A resolution structure of human fatty acid synthase thioesterase domain reported here is comprised of two dissimilar subdomains, A and B. The smaller subdomain B is composed entirely of alpha-helices arranged in an atypical fold, whereas the A subdomain is a variation of the alpha/beta hydrolase fold. The structure revealed the presence of a hydrophobic groove with a distal pocket at the interface of the two subdomains, which constitutes the candidate substrate binding site. The length and largely hydrophobic nature of the groove and pocket are consistent with the high selectivity of the thioesterase for palmitoyl acyl substrate. The structure also set the identity of the Asp residue of the catalytic triad of Ser, His, and Asp located in subdomain A at the proximal end of the groove.

Amino Acid Sequence↗

PubMed2004

Structure of the bifunctional and Golgi-associated formiminotransferase cyclodeaminase octamer.

Mammalian formiminotransferase cyclodeaminase (FTCD), a 0.5 million Dalton homo-octameric enzyme, plays important roles in coupling histidine catabolism with folate metabolism and integrating the Golgi complex with the vimentin intermediate filament cytoskeleton. It is also linked to two human diseases, autoimmune hepatitis and glutamate formiminotransferase deficiency. Determination of the FTCD structure by X-ray crystallography and electron cryomicroscopy revealed that the eight subunits, each composed of distinct FT and CD domains, are arranged like a square doughnut. A key finding indicates that coupling of three subunits governs the octamer-dependent sequential enzyme activities, including channeling of intermediate and conformational change. The structure further shed light on the molecular nature of two strong antigenic determinants of FTCD recognized by autoantibodies from patients with autoimmune hepatitis and on the binding of thin vimentin filaments to the FTCD octamer.

Amino Acid Sequence↗

PubMed2003

The crystal structure of the gene targeting homing endonuclease I-SceI reveals the origins of its target site specificity.

The I-SceI homing endonuclease enhances gene targeting by introducing double-strand breaks at specific chromosomal loci, thereby increasing the recombination frequency. Here, we report the crystal structure of the enzyme complexed to its DNA substrate and Ca(2+) determined at 2.25A resolution. The structure shows the prototypical beta-saddle of LAGLIDADG homing endonucleases that is contributed by two pseudo-symmetric domains. The high specificity of I-SceI is explained by the large number of protein-DNA contacts, many that are made by a long beta-hairpin loop that reaches into the major groove of the DNA. The DNA minor groove is compressed at the catalytic center, bringing the two scissile phosphodiester bonds into close proximity. The protein-Ca(2+)-DNA structure shows the protein bound to its DNA substrate in a pre-reactive state that is defined by the presence of two asymmetric active sites, one of which appears poised to first cleave the DNA bottom strand.

Base Sequence↗

PubMed2003

Structural basis of peptide-carbohydrate mimicry in an antibody-combining site.

The structure of a complex between the Fab fragment of the antibody (SYA/J6) specific for the cell surface O-antigen polysaccharide of the pathogen Shigella flexneri Y and an octapeptide (Met-Asp-Trp-Asn-Met-His-Ala-Ala), a functional mimic of the O-antigen, has been determined at 1.8-A resolution. Comparison of the structure with that of the complex with the pentasaccharide antigen [-->2)-alpha-L-Rha-(1-->2)-alpha-L-Rha-(1-->3)-alpha-L-Rha-(1-->3)-beta-D-GlcNAc-(1-->2)-alpha-L-Rha-(1-->] reveals the molecular recognition process by which a peptide mimics a carbohydrate in binding to an antibody. The binding modes of the two ligands differ considerably. Octapeptide binding complements the shape of the combining site groove much better than pentasaccharide binding. Moreover, the peptide makes a much greater number of contacts (126), which are mostly van der Waals interactions, with the Fab than the saccharide (74). An unusual feature is also the involvement of 12 water molecules in mediating hydrogen bonds between residues within the peptide or of the peptide and Fab. Despite better shape complementarity and greater number of contacts, the octapeptide binds with an affinity (KA = 2.5 x 10(5) M-1, measured by calorimetry) only approximately 2-fold tighter than the pentasaccharide. The structural results are relevant to the design of peptide mimetics with improved affinity for use as vaccines.

Animals↗

PubMed2003

Insertion of an N7-methylguanine mRNA cap between two coplanar aromatic residues of a cap-binding protein is fast and selective for a positively charged cap.

The N7-methylated guanosine (m7G) cap structure, which is found at the 5' ends of mature eukaryotic mRNAs, is critical to a myriad of biological processes. The twenty structures of complexes of cap nucleosides and nucleotides and methylated bases with the vaccinia virus VP39, a cap-specific RNA 2'-O-methyltransferase, which we have determined previously, have revealed the atomic basis of cap binding. The precise insertion and tight fitting of the m7Gua moiety of the cap between two parallel aromatic residues that are spaced only 6.8 A apart governs the high specificity of binding. Here we report the investigation of the reaction mechanism of VP39 with three capped ligands (m7G, m7GpppG, and m7GpppGA3) by fluorescence stopped-flow technique. Cap binding is a simple one-step mechanism with very fast association rate constant (approximately 10(7) M-1 s-1). Moreover, the pH dependence on the association rate constant of m7G binding indicates that only the positively charged keto tautomer of the cap is recognized and bound. The association and dissociation rate constants and affinity constants of the three ligands do not vary greatly, demonstrating that binding is achieved almost entirely by the interactions of m7Gua with two aromatic residues in a cation-pi sandwich.

Amino Acids, Aromatic↗

PubMed2003

Architecture of the herpes simplex virus major capsid protein derived from structural bioinformatics.

The dispositions of 39 alpha helices of greater than 2.5 turns and four beta sheets in the major capsid protein (VP5, 149 kDa) of herpes simplex virus type 1 were identified by computational and visualization analysis from the 8.5A electron cryomicroscopy structure of the whole capsid. The assignment of helices in the VP5 upper domain was validated by comparison with the recently determined crystal structure of this region. Analysis of the spatial arrangement of helices in the middle domain of VP5 revealed that the organization of a tightly associated bundle of ten helices closely resembled that of a domain fold found in the annexin family of proteins. Structure-based sequence searches suggested that sequences in both the N and C-terminal portions of the VP5 sequence contribute to this domain. The long helices seen in the floor domain of VP5 form an interconnected network within and across capsomeres. The combined structural and sequence-based informatics has led to an architectural model of VP5. This model placed in the context of the capsid provides insights into the strategies used to achieve viral capsid stability.

Amino Acid Motifs↗

PubMed2003

Structure of the herpesvirus major capsid protein.

Herpes simplex virus-1 (HSV-1) virions are large, complex enveloped particles containing a proteinaceous tegument layer connected to an icosahedral capsid. The major capsid protein, VP5 (149 kDa), makes up both types of capsomere, pentons and hexons. Limited trypsin digestion of VP5 identified a single stable 65 kDa fragment which represents a proposed protein folding nucleus. We report the 2.9 A crystal structure of this fragment and its modeling into an 8.5 A resolution electron cryomicroscopy map of the HSV-1 capsid. The structure, the first for any capsid protein from Herpesviridae, revealed a novel fold, placing herpesviruses outside any of the structurally linked viral groupings. Alterations in the geometrical arrangements of the VP5 subunits in the capsomeres exposes different residues, resulting in the differential association of the tegument and VP26 with the pentons and hexons, respectively. The rearrangements of VP5 subunits required to form both pentavalent and hexavalent capsomeres result in structures that exhibit very different electrostatic properties. These differences may mediate the binding and release of other structural proteins during capsid maturation.

Capsid Proteins↗

PubMed2003

A tweezers-like motion of the ATP-binding cassette dimer in an ABC transport cycle.

The ATPase components of ATP binding cassette (ABC) transporters power the transporters by binding and hydrolyzing ATP. Major conformational changes of an ATPase are revealed by crystal structures of MalK, the ATPase subunit of the maltose transporter from Escherichia coli, in three different dimeric configurations. While other nucleotide binding domains or subunits display low affinity for each other in the absence of the transmembrane segments, the MalK dimer is stabilized through interactions of the additional C-terminal domains. In the two nucleotide-free structures, the N-terminal nucleotide binding domains are separated to differing degrees, and the dimer is maintained through contacts of the C-terminal regulatory domains. In the ATP-bound form, the nucleotide binding domains make contact and two ATPs lie buried along the dimer interface. The two nucleotide binding domains of the dimer open and close like a pair of tweezers, suggesting a regulatory mechanism for ATPase activity that may be tightly coupled to translocation.

ATP-Binding Cassette Transporters↗

PubMed2003

A closed compact structure of native Ca(2+)-calmodulin.

Calmodulin has been a subject of intense scrutiny since its discovery because of its unusual properties in regulating the functions of about 100 diverse target enzymes and structural proteins. The original and to date only crystal conformation of native eukaryotic Ca(2+)-calmodulin (Ca(2+)-CaM) is a very extended molecule with two widely separated globular domains linked by an exposed long helix. Here we report the 1.7 A X-ray structure of a new native Ca(2+)-CaM that is in a compact ellipsoidal conformation and shows a sharp bend in the linker helix and a more contracted N-terminal domain. This conformation may offer advantages for recognition of kinase-type calmodulin targets or small organic molecule drugs.

Animals↗

PubMed2003

Crystal structure of M tuberculosis ABC phosphate transport receptor: specificity and charge compensation dominated by ion-dipole interactions.

The 2.16 A structure of the phosphate-bound PstS-1, the primary extracellular receptor for the ABC phosphate transporter and immunodominant species-specific antigen of Mycobacterium tuberculosis, has been determined. The phosphate, completely engulfed in the cleft between two domains, is bound by 13 hydrogen bonds, 11 of which are formed with NH and OH dipolar donor groups. The further presence of two acidic residues, which serve as acceptors of the protonated phosphate, is key to conferring stringent specificity. The ion-dipole interactions between the phosphate and dipolar groups compensate the ligand's isolated negative charges. Moreover, the surprise finding that the electrostatic surface in and around the cleft is intensely negative demonstrates the power of ion-dipole interactions in anion binding and electrostatic balance. Additional functional features include both the flexible N-terminal segment that tethers PstS-1 on the cell surface and the hinge between the two domains, which should facilitate snaring the phosphate in the medium.

ATP-Binding Cassette Transporters↗

PubMed2002

Molecular recognition of oligosaccharide epitopes by a monoclonal Fab specific for Shigella flexneri Y lipopolysaccharide: X-ray structures and thermodynamics.

The antigenic recognition of Shigella flexneri O-polysaccharide, which consists of a repeating unit ABCD [-->2)-alpha-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->3)-beta-D-GlcpNAc-(1-->], by the monoclonal antibody SYA/J6 (IgG3, kappa) has been investigated by crystallographic analysis of the Fab domain and its two complexes with two antigen segments (a pentasaccharide Rha A-Rha B-Rha C-GlcNAc D-Rha A' and a modified trisaccharide Rha B-Rha C-GlcNAc D in which Rha C* is missing a C2-OH group). These complex structures, the first for a Fab specific for a periodic linear heteropolysaccharide, reveal a binding site groove (between the V(H) and V(L) domains) that makes polar and nonpolar contacts with all the sugar residues of the pentasaccharide. Both main-chain and side-chain atoms of the Fab are used in ligand binding. The charged side chain of Glu H50 of CDR H2 forms crucial hydrogen bonds to GlcNAc of the oligosaccharides. The modified trisaccharide is more buried and fits more snugly than the pentasaccharide. It also makes as many contacts (approximately 75) with the Fab as the pentasaccharide, including the same number of hydrogen bonds (eight, with four being identical). It is further engaged in more hydrophobic interactions than the pentasaccharide. These three features favorable to trisaccharide binding are consistent with the observation of a tighter complex with the trisaccharide than the pentasaccharide. Thermodynamic data demonstrate that the native tri- to pentasaccharides have free energies of binding in the range of 6.8-7.4 kcal mol(-1), and all but one of the hydrogen bonds to individual hydroxyl groups provide no more than approximately 0.7 kcal mol(-1). They further indicate that hydrophobic interactions make significant contributions to binding and, as the native epitope becomes larger across the tri-, tetra-, pentasaccharide series, entropy contributions to the free energy become dominant.

Antibodies, Monoclonal↗