Biomedical subjects

Numan Oezguen

Publications and source records attributed to Numan Oezguen.

8 recordsLinked to original sources

PubMed2006

Novel, structure-based mechanism for uridylylation of the genome-linked peptide (VPg) of picornaviruses.

The VPg peptide, which is found in poliovirus infected cells either covalently bound to the 5'-end of both plus and minus strand viral RNA, or in a uridylylated free form, is essential for picornavirus replication. Combining experimental structure and mutation results with molecular modeling suggests a new mechanism for VPg uridylylation, which assigns an additional function, that of scaffold, to the polymerase. The polarity of the NMR structure of VPg is complementary to the binding site on the surface of poliovirus polymerase determined previously by mutagenesis. Docking VPg at this position places the reactive tyrosinate close to the 5'-end of Poly(A)7 RNA when this is bound with its 3'-end in the active site of the polymerase. The triphosphate tail of a UTP moiety, base paired with the 5'-end of the RNA, projects back over the Tyr3-OH and is held in position by conserved positively charged side-chains of VPg. Other conserved residues mediate binding to the polymerase surface and serve as ligands for metal ion catalyzed transphosphorylation. Additional viral proteins or a second polymerase molecule may aid in stabilizing the components of the reaction. In the model complex, VPg can direct its own uridylylation before entering the polymerase active site.

Amino Acid Sequence↗

PubMed2006

NMR structure of the viral peptide linked to the genome (VPg) of poliovirus.

VPgs are essential for replication of picornaviruses, which cause diseases such as poliomyelitis, foot and mouth disease, and the common cold. VPg in infected cells is covalently linked to the 5' end of the viral RNA, or, in a uridylylated form, free in the cytoplasm. We show here the first solution structure for a picornaviral VPg, that of the 22-residue peptide from poliovirus serotype 1. VPg in buffer is inherently flexible, but a single conformer was obtained by adding trimethylamine N-oxide (TMAO). TMAO had only minor effects on the TOCSY spectrum. However, it increased the amount of structured peptide, as indicated by more peaks in the NOESY spectrum and an up to 300% increase in the ratio of normalized NOE cross peak intensities to that in buffer. The data for VPg in TMAO yielded a well defined structure bundle with 0.6 A RMSD (versus 6.6 A in buffer alone), with 10-30 unambiguous constraints per residue. The structure consists of a large loop region from residues 1 to 14, from which the reactive tyrosinate projects outward, and a C-terminal helix from residues 18 to 21 that aligns the sidechains of conserved residues on one face. The structure has a stable docking position at an area on the poliovirus polymerase crystal structure identified as a VPg binding site by mutagenesis studies. Further, UTP and ATP dock in a base-specific manner to the reactive face of VPg, held in place by residues conserved in all picornavirus VPgs.

Adenosine Triphosphate↗

PubMed2005

Molego-based definition of the architecture and specificity of metal-binding sites.

Decomposing proteins into "molegos," building blocks that are conserved in sequence and 3D-structure, can identify functional elements. To demonstrate the specificity of the decomposition method, the PCPMer program suite was used to numerically define physical chemical property motifs corresponding to the molegos that make up the metal-containing active sites of three distinct enzyme families, from the dimetallic phosphatases, DNase 1 related nucleases/phosphatases, and dioxygenases. All three superfamilies bind metal ions in a beta-strand core region but differ in the number and type of ions needed for activity. The motifs were then used to automatically identify proteins in the ASTRAL40 database that contained similar motifs. The proteins with the highest PCPMer score in the database were primarily metal-binding enzymes that were related in function to those in the alignment used to generate the PCPMer motif lists. The proteins that contained motifs similar to the dioxygenases differed from those found with PCP-motifs for phosphatases and nucleases. Relatively few metal-binding enzymes were detected when the search was done with PCP-motifs defined for interleukin-1 related proteins, which have a beta-strand core but do not bind metal ions. While the box architecture was constant in each superfamily, the specificity for the metal ion preferred for enzymatic activity is determined by the pattern of carbonyl, hydroxyl or imadazole groups in key positions in the molegos. These results have implications for the design of metal-binding enzymes, and illustrate the ability of the PCPMer approach to distinguish, at the sequence level, structural and functional elements.

Binding Sites↗

PubMed2005

Nearby clusters of hemagglutinin residues sustain SLAM-dependent canine distemper virus entry in peripheral blood mononuclear cells.

Signaling lymphocytic activation molecule (SLAM, CD150) is the universal morbillivirus receptor. Based on the identification of measles virus (MV) hemagglutinin (H) amino acids supporting human SLAM-dependent cell entry, we mutated canine distemper virus (CDV) H and identified residues necessary for efficient canine SLAM-dependent membrane fusion. These residues are located in two nearby clusters in a new CDV H structural model. To completely abolish SLAM-dependent fusion, combinations of mutations were necessary. We rescued a SLAM-blind recombinant CDV with six mutations that did not infect ferret peripheral blood mononuclear cells while retaining full infectivity in epithelial cells.

Amino Acid Sequence↗

PubMed2004

Effects of backbone contacts 3' to the abasic site on the cleavage and the product binding by human apurinic/apyrimidinic endonuclease (APE1).

The mammalian apurinic/apyrimidinic (AP) endonuclease (APE1) is a multifunctional protein that plays essential roles in DNA repair and gene regulation. We decomposed the APEs into 12 blocks of highly conserved sequence and structure (molegos). This analysis suggested that residues in molegos common to all APEs, but not to the less specific nuclease, DNase I, would dictate enhanced binding to damaged DNA. To test this hypothesis, alanine was substituted for N226 and N229, which form hydrogen bonds to the DNA backbone 3' of the AP sites in crystal structures of the APE1/DNA complex. While the cleavage rate at AP sites of both N226A and N229A mutants increased, their ability to bind to damaged DNA decreased. The ability of a double mutant (N226A/N229A) to bind damaged DNA was further decreased, while the V(max) was almost identical to that of the wild-type APE1. A double mutant at N226 and R177, a residue that binds to the same phosphate as N229, had a significantly decreased activity and substrate binding. As the affinity for product DNA was decreased in all the mutants, the enhanced reaction rate of the single mutants could be due to alleviation of product inhibition of the enzyme. We conclude that hydrogen bonds to phosphate groups 3' to the cleavage site is essential for APE1's binding to the product DNA, which may be necessary for efficient functioning of the base excision repair pathway. The results indicate that the molego analysis can aid in the redesign of proteins with altered binding affinity and activity.

Alanine↗

PubMed2004

Using property based sequence motifs and 3D modeling to determine structure and functional regions of proteins.

Homology modeling has become an essential tool for studying proteins that are targets for medical drug design. This paper describes the approach we developed that combines sequence decomposition techniques with distance geometry algorithms for homology modeling to determine functionally important regions of proteins. We show here the application of these techniques to targets of medical interest chosen from those included in the CASP5 (Critical Assessment of Techniques for Protein Structure Prediction) competition, including the dihydroneopterin aldolase from Mycobacterium tuberculosis, RNase III of Thermobacteria maritima, and the NO-transporter nitrophorin from saliva of the bedbug Cimex lectularius. Physical chemical property (PCP) motifs, identified in aligned sequences with our MASIA program, can be used to select among different alignments returned by fold recognition servers. They can also be used to suggest functions for hypothetical proteins, as we illustrate for target T188. Once a suitable alignment has been made with the template, our modeling suite MPACK generates a series of possible models. The models can then be selected according to their match in areas known to be conserved in protein families. Alignments based on motifs can improve the structural matching of residues in the active site. The quality of the local structure of our 3D models near active sites or epitopes makes them useful aids for drug and vaccine design. Further, the PCP motif approach, when combined with a structural filter, can be a potent way to detect areas involved in activity and to suggest function for novel genome sequences.

Aldehyde-Lyases↗

PubMed2002

Membrane-protein interactions contribute to efficient 27-hydroxylation of cholesterol by mitochondrial cytochrome P450 27A1.

Mitochondrial cytochrome P450 27A1 (P450 27A1) catalyzes 27-hydroxylation of cholesterol, the first step in the alternative bile acid biosynthetic pathway. Although several crystal structures of P450s are known, no structural information is available for the mammalian, membrane-bound enzymes involved in the removal of cholesterol from the body. We prepared a three-dimensional model of P450 27A1 based on the structure of P450 BM-3. Conservative and non-conservative mutations were introduced at hydrophobic and positively charged residues in the putative F-G loop and the adjacent helix G (positions 219-237). Subcellular distribution of the mutant P450s expressed in Escherichia coli was used as a measure of membrane-protein interactions. Conservative substitutions of residues located on the surface, according to our model, L219V, L219I, Y220F, F223Y, L224I, R229K, V231L, F234Y, K236R, and R237K, weakened the association of the mutant P450s with the membrane and led to the appearance of up to 21% of P450 27A1 in the bacterial cytosol. It is likely that the mutated side chains are involved in binding to membrane phospholipids. Substitutions in the F-G loop did not significantly affect the K(m) value for cholesterol hydroxylation. However, non-conservative mutants, L219N, Y220A, Y220S, F223A, K226R, and R229A, had significantly impaired catalytic properties, indicating strict requirements for the size and polarity of the side chains at these positions for the catalysis. The results provide insight into the membrane topology of mitochondrial P450s and indicate the importance of membrane-protein interactions in the efficiency of reactions catalyzed by P450 27A1.

Amino Acid Sequence↗

PubMed2002

Automated assignment and 3D structure calculations using combinations of 2D homonuclear and 3D heteronuclear NOESY spectra.

The NOAH/DIAMOD suite uses feedback filtering and self-correcting distance geometry to generate 3D structures from unassigned NOESY spectra. In this study we determined the minimum set of experiments needed to generate a high quality structure bundle. Different combinations of 3D 15N-edited, 13C-edited HSQC-NOESY and 2D homonuclear 1H-1H NOESY spectra of the 77 amino acid protein, myeloid progenitor inhibitory factor-1 (MPIF-1) were used as input for NOAH/DIAMOD calculations. The quality of the assignments of NOESY cross peaks and the accuracy of the automatically generated 3D structures were compared to those obtained with a conventional manual procedure. Combining data from two types of experiments synergistically increased the number of peaks assigned unambiguously in both individual spectra. As a general trend for the accuracy of the structures we observed structural variations in the backbone fold of the final structures of about 2 A for single spectral data, of 1 A to 1.5 A for double spectral data, and of 0.6 A for triple spectral data sets. The quality of the assignments and 3D structures from the optimal data using all three spectra were similar to those obtained from traditional assignment methods with structural variations within the bundle of 0.6 A and 1.3 A for backbone and heavy atoms, respectively. Almost all constraints (97%) of the automatic NOESY cross peak assignments were cross compatible with the structures from the conventional manual assignment procedure, and an even larger proportion (99%) of the manually derived constraints were compatible with the automatically determined 3D structures. The two mean structures determined by both methods differed only by 1.3 A rmsd for the backbone atoms in the well-defined regions of the protein. Thus NOAD/DIAMOD analysis of spectra from labeled proteins provides a reliable method for high throughput analysis of genomic targets.

Animals↗