Ibrahim Moustafa - Academia.edu (original) (raw)

Papers by Ibrahim Moustafa

The Journal of Biological Chemistry, 2008

Picornaviruses have a peptide termed VPg covalently linked to the 5-end of the genome. Attachment... more Picornaviruses have a peptide termed VPg covalently linked to the 5-end of the genome. Attachment of VPg to the genome occurs in at least two steps. First, Tyr-3 of VPg, or some precursor thereof, is used as a primer by the viral RNA-dependent RNA polymerase, 3Dpol, to produce VPg-pUpU. Second, VPg-pUpU is used as a primer to produce full-length genomic RNA. Production of VPg-pUpU is templated by a single adenylate residue located in the loop of an RNA stem-loop structure termed oriI by using a slide-back mechanism. Recruitment of 3Dpol to and its stability on oriI have been suggested to require an interaction between the back of the thumb subdomain of 3Dpol and an undefined region of the 3C domain of viral protein 3CD. We have performed surface acidic-to-alanine-scanning mutagenesis of 3C to identify the surface of 3C with which 3Dpol interacts. This analysis identified numerous viable poliovirus mutants with reduced growth kinetics that correlated to reduced kinetics of RNA synthesis that was attributable to a change in VPg-pUpU production. Importantly, these 3C derivatives were all capable of binding to oriI as well as wild-type 3C. Synthetic lethality was observed for these mutants when placed in the context of a poliovirus mutant containing 3Dpol-R455A, a residue on the back of the thumb required for VPg uridylylation. These data were used to guide molecular docking of the structures for a poliovirus 3C dimer and 3Dpol, leading to a structural model for the 3C 2 -3Dpol complex that extrapolates well to all picornaviruses.

Viruses, 2015

The genomes of RNA viruses are relatively small. To overcome the small-size limitation, RNA virus... more The genomes of RNA viruses are relatively small. To overcome the small-size limitation, RNA viruses assign distinct functions to the processed viral proteins and their precursors. This is exemplified by poliovirus 3CD protein. 3C protein is a protease and RNA-binding protein. 3D protein is an RNA-dependent RNA polymerase (RdRp). 3CD exhibits unique protease and RNA-binding activities relative to 3C and is devoid of RdRp activity. The origin of these differences is unclear, since crystal structure of 3CD revealed &amp;quot;beads-on-a-string&amp;quot; structure with no significant structural differences compared to the fully processed proteins. We performed molecular dynamics (MD) simulations on 3CD to investigate its conformational dynamics. A compact conformation of 3CD was observed that was substantially different from that shown crystallographically. This new conformation explained the unique properties of 3CD relative to the individual proteins. Interestingly, simulations of mutant 3CD showed altered interface. Additionally, accelerated MD simulations uncovered a conformational ensemble of 3CD. When we elucidated the 3CD conformations in solution using small-angle X-ray scattering (SAXS) experiments a range of conformations from extended to compact was revealed, validating the MD simulations. The existence of conformational ensemble of 3CD could be viewed as a way to expand the poliovirus proteome, an observation that may extend to other viruses.

PLoS pathogens, 2015

High rates of error-prone replication result in the rapid accumulation of genetic diversity of RN... more High rates of error-prone replication result in the rapid accumulation of genetic diversity of RNA viruses. Recent studies suggest that mutation rates are selected for optimal viral fitness and that modest variations in replicase fidelity may be associated with viral attenuation. Arthropod-borne viruses (arboviruses) are unique in their requirement for host cycling and may necessitate substantial genetic and phenotypic plasticity. In order to more thoroughly investigate the correlates, mechanisms and consequences of arbovirus fidelity, we selected fidelity variants of West Nile virus (WNV; Flaviviridae, Flavivirus) utilizing selection in the presence of a mutagen. We identified two mutations in the WNV RNA-dependent RNA polymerase associated with increased fidelity, V793I and G806R, and a single mutation in the WNV methyltransferase, T248I, associated with decreased fidelity. Both deep-sequencing and in vitro biochemical assays confirmed strain-specific differences in both fidelity ...

Journal of molecular biology, Jan 15, 2006

Nucleic Acids and Molecular Biology, 2013

The RNA-dependent RNA polymerase (RdRp) is responsible for replicating the genomes of RNA viruses... more The RNA-dependent RNA polymerase (RdRp) is responsible for replicating the genomes of RNA viruses. The overall structure and function of RdRps is similar to other nucleic acid polymerases, although some RdRps employ unique initiation mechanisms. Recent biophysical studies indicate that the internal motions of RdRps, and other nucleic acid polymerases, are critical for their catalytic function and fidelity. In particular, these studies have suggested that the closing of the active site in preparation for catalysis involves the movement of the motif-D loop to help reposition a highly conserved lysine, enabling this residue to act as a general acid to protonate the pyrophosphate leaving group. Binding of incorrect nucleoside triphosphate does not induce the same structural changes in the motif-D loop, indicating a role for this loop in nucleotide discrimination. Indeed, substitution at the motif-D lysine increases polymerase fidelity and, intriguingly, decreases viral pathogenesis. The highly conserved nature of this lysine thus suggests a universal mechanism for rational vaccine design based on generating variants at this position. Moreover, substitutions elsewhere in the RdRp structure, including those remote from the active site, likewise lead to changes in polymerase fidelity and decrease viral pathogenesis. In these cases, the amino acid substitutions alter internal protein motions (including those in the motif-D loop) without substantially affecting the polymerase structure. A picture emerges in which RdRps and other nucleic acid polymerases can be viewed as "small world" networks of amino acids; communication pathways connect from the surface of the protein all the way to the catalytic center. These networks can be impacted by amino acid substitutions, inhibitor binding, and/or binding of accessory replication proteins to regulate RdRp catalysis and fidelity.

The Journal of Physical Chemistry B, 2012

Proceedings of the National Academy of Sciences, 2012

Cell-based studies support the existence of two promoters on the heavy strand of mtDNA: heavy-str... more Cell-based studies support the existence of two promoters on the heavy strand of mtDNA: heavy-strand promoter 1 (HSP1) and HSP2. However, transcription from HSP2 has been reported only once in a cell-free system, and never when recombinant proteins have been used. Here, we document transcription from HSP2 using an in vitro system of defined composition. An oligonucleotide template representing positions 596-685 of mtDNA was sufficient to observe transcription by the human mtRNA polymerase (POLRMT) that was absolutely dependent on mitochondrial transcription factor B2 (TFB2M). POLRMT/TFB2M-dependent transcription was inhibited by concentrations of mitochondrial transcription factor A (TFAM) stoichiometric with the transcription template, a condition that activates transcription from the light-strand promoter (LSP) in vitro. Domains of TFAM required for LSP activation were also required for HSP2 repression, whereas other mtDNA binding proteins failed to alter transcriptional output. Binding sites for TFAM were located on both sides of the start site of transcription from HSP2, suggesting that TFAM binding interferes with POLRMT and/or TFB2M binding. Consistent with a competitive binding model for TFAM repression of HSP2, the impact of TFAM concentration on HSP2 transcription was diminished by elevating the POLRMT and TFB2M concentrations. In the context of our previous studies of LSP and HSP1, it is now clear that three promoters exist in human mtDNA. Each promoter has a unique requirement for and/or response to the level of TFAM present, thus implying far greater complexity in the regulation of mammalian mitochondrial transcription than recognized to date. mitochondria | gene expression | initiation | gel-shift assay | footprinting

Nature Structural & Molecular Biology, 2009

Nucleic acid polymerases catalyze the formation of DNA or RNA from nucleoside-triphosphate precur... more Nucleic acid polymerases catalyze the formation of DNA or RNA from nucleoside-triphosphate precursors. Amino acid residues in the active site of polymerases are thought to contribute only indirectly to catalysis by serving as ligands for the two divalent cations that are required for activity or substrate binding. Two proton-transfer reactions are necessary for polymerase-catalyzed nucleotidyl transfer: deprotonation of the 3¢-hydroxyl nucleophile and protonation of the pyrophosphate leaving group. Using model enzymes representing all four classes of nucleic acid polymerases, we show that the proton donor to pyrophosphate is an active-site amino acid residue. The use of general acid catalysis by polymerases extends the mechanism of nucleotidyl transfer beyond that of the well-established two-metal-ion mechanism. The existence of an active-site residue that regulates polymerase catalysis may permit manipulation of viral polymerase replication speed and/or fidelity for virus attenuation and vaccine development.

Nature Chemical Biology, 2006

Hydrogen atoms are a vital component of enzyme structure and function 1-4 . In recent years, atom... more Hydrogen atoms are a vital component of enzyme structure and function 1-4 . In recent years, atomic resolution crystallography (Z1.2 Å ) has been successfully used to investigate the role of the hydrogen atom in enzymatic catalysis [5] . Here, atomic resolution crystallography was used to study the effect of pH on cholesterol oxidase from Streptomyces sp., a flavoenzyme oxidoreductase. Crystallographic observations of the anionic oxidized flavin cofactor at basic pH are consistent with the UV-visible absorption profile of the enzyme and readily explain the reversible pH-dependent loss of oxidation activity. Furthermore, a hydrogen atom, positioned at an unusually short distance from the main chain carbonyl oxygen of Met122 at high pH, was observed, suggesting a previously unknown mechanism of cofactor stabilization. This study shows how a redox active site responds to changes in the enzyme's environment and how these changes are able to influence the mechanism of enzymatic catalysis.

Journal of Virology, 2002

We recently reported the first crystal structure of a paramyxovirus hemagglutinin-neuraminidase (... more We recently reported the first crystal structure of a paramyxovirus hemagglutinin-neuraminidase (HN) from Newcastle disease virus. This multifunctional protein is responsible for binding to cellular sialyl-glycoconjugate receptors, promotion of fusion through interaction with the second viral surface fusion (F) glycoprotein, and processing progeny virions by removal of sialic acid from newly synthesized viral coat proteins. Our structural studies suggest that HN possesses a single sialic acid recognition site that can be switched between being a binding site and a catalytic site. Here we examine the effect of mutation of several conserved amino acids around the binding site on the hemagglutination, neuraminidase, and fusion functions of HN. Most mutations around the binding site result in loss of neuraminidase activity, whereas the effect on receptor binding is more variable. Residues E401, R416, and Y526 appear to be key for receptor binding. The increase in fusion promotion seen in some mutants that lack receptor binding activity presents a conundrum. We propose that in these cases HN may be switched into a fusion-promoting state through a series of conformational changes that propagate from the sialic acid binding site through to the HN dimer interface. These results further support the single-site model and suggest certain residues to be important for the triggering of fusion.

Journal of Virology, 2008

Replication of picornaviral genomes requires recognition of at least three cis-acting replication... more Replication of picornaviral genomes requires recognition of at least three cis-acting replication elements: oriL, oriI, and oriR. Although these elements lack an obvious consensus sequence or structure, they are all recognized by the virus-encoded 3C protein. We have studied the poliovirus 3C-oriI interaction in order to begin to decipher the code of RNA recognition by picornaviral 3C proteins. oriI is a stem-loop structure that serves as the template for uridylylation of the peptide primer VPg by the viral RNA-dependent RNA polymerase. In this report, we have used nuclear magnetic resonance (NMR) techniques to study 3C alone and in complex with two single-stranded RNA oligonucleotides derived from the oriI stem. The 1 H-15 N spectra of 3C recorded in the presence of these RNAs revealed site-specific chemical shift perturbations. Residues that exhibit significant perturbations are primarily localized in the amino terminus and in a highly conserved loop between residues 81 and 89. In general, the RNA-binding site defined in this study is consistent with predictions based on biochemical and mutagenesis studies. Although some residues implicated in RNA binding by previous studies are perturbed in the 3C-RNA complex reported here, many are unique. These studies provide unique site-specific insight into residues of 3C that interact with RNA and set the stage for detailed structural investigation of the 3C-RNA complex by NMR. Interpretation of our results in the context of an intact oriI provides insight into the architecture of the picornavirus VPg uridylylation complex.

Journal of Molecular Biology, 2006

L-Amino acid oxidase is a dimeric glycosylated flavoenzyme, a major constituent of the venom-from... more L-Amino acid oxidase is a dimeric glycosylated flavoenzyme, a major constituent of the venom-from the snake Calloselasma rhodostoma. The enzyme exhibits apoptosis inducing effects as well as antibacterial and anti-HIV activities. The structure of L-amino acid oxidase with its substrate (L-phenylalanine) has been refined to a resolution of 1.8 Å. The complex structure reveals the substrate bound to the reduced flavin (FAD red ). Alternative conformations for the key residues His223 and Arg322 are evident, suggesting a dynamic active site. Furthermore, conformational changes are apparent for the isoalloxazine ring; the three-ring system exhibits more bending around the N5-N10 axis compared to the oxidized flavin. The implications of the observed dynamics on the mechanism of catalysis are discussed. Inspection of buried surfaces in the enzyme reveals a Y-shaped channel system extending from the external surface of the protein to the active site. One portion of this channel may serve as the entry path for O 2 during the oxidative half-reaction. The second region, separated from the proposed O 2 channel by the N terminus (residues 8-16) of the protein, may play a role in H 2 O 2 release. Interestingly, the latter portion of the channel would direct the H 2 O 2 product to the exterior surface of the protein, near the glycan moiety, thought to anchor the enzyme to the host cell. This channel location may explain the ability of the enzyme to localize H 2 O 2 to the targeted cell and thus induce the apoptotic effect.

Journal of Molecular Biology, 2011

The viral RNA-dependent RNA polymerase (RdRp) is essential for multiplication of all RNA viruses.... more The viral RNA-dependent RNA polymerase (RdRp) is essential for multiplication of all RNA viruses. The sequence diversity of an RNA virus population contributes to its ability to infect the host. This diversity emanates from errors made by the RdRp during RNA synthesis. The physical basis for RdRp fidelity is unclear but is linked to conformational changes occurring during the nucleotide-addition cycle. To understand RdRp dynamics that might influence RdRp function, we have analyzed all-atom molecular dynamics simulations on the nanosecond timescale of four RdRps from the picornavirus family that exhibit 30-74% sequence identity. Principal component analysis showed that the major motions observed during the simulations derived from conserved structural motifs and regions of known function. The dynamics of residues participating in the same biochemical property, for example, RNA binding, nucleotide binding or catalysis, were correlated even when spatially distant on the RdRp structure. The conserved and correlated dynamics of functional structural elements suggest coevolution of dynamics with structure and function of the RdRp. Crystal structures of all picornavirus RdRps exhibit a template-nascent RNA duplex channel too small to fully accommodate duplex RNA. Simulations revealed opening and closing motions of the RNA and nucleoside triphosphate channels, which might be relevant to nucleoside triphosphate entry, inorganic pyrophosphate exit and translocation. A role for nanosecond timescale dynamics in RdRp fidelity is supported by the altered dynamics of the high-fidelity G64S derivative of PV RdRp relative to wild-type enzyme.

Journal of Hepatology, 2000

Journal of Hepatology, 2002

Category 2: Cirrhosis and its complications, pathophysiology and clinical aspects 201 Material: 4... more Category 2: Cirrhosis and its complications, pathophysiology and clinical aspects 201 Material: 42 patients with cirrhosis (16 with severe PHG, 14 with mild PHG and 12 without PHG) and 13 healthy subjects were included in the study. Endoscopic gastric biopsies were examined for microvascular changes by morphometric analysis of sections stained with vascular endothelial cell adhesion molecule and for epithelial proliferation using immunostaining with proliferating cell nuclear antigen (PCNA). Levels of serum nitrite and nitrate and plasma endothelin-1 were measured. Results: Biopsies from gastric fundus and corpus showed a significant increase in the total vascular area, number of vessels and vessel wall thickness and a significant decrease in the PCNA-labelling index in cirrhotic patients with PHG compared with those without PHG and healthy subjects and in patients with severe PHG compared with those with mild PHG (P < 0.05). In antral mucosa, these changes were only evident in patients with severe PHG (P < 0.05). Serum nitrite and nitrate levels showed a significant stepwise increase in cirrhotic patients without PHG through patients with mild PHG to patients with severe PHG compared with healthy subjects (P < 0.05). Plasma ET-1 concentration was significantly higher in patients with cirrhosis regardless of the presence or severity of PHG than in healthy subjects (P < 0.05). Serum nitrite and nitrate levels showed a direct correlation with the gastric morphometric microvascular changes and an inverse correlation with the PCNA-labelling index of the fundic and corporeal mucosa in cirrhotic patients (P < 0.01). Conclusions: Excessive production of NO (but not of ET-1) in patients with cirrhosis is likely to be involved in the vasodilatation of gastric vessels and the inhibition of gastric epithelial proliferation in PHG.

Journal of Biological Chemistry, 2008

Journal of Biological Chemistry, 2010

Hepatitis C virus non-structural protein 3 contains a serine protease and an RNA helicase. Protea... more Hepatitis C virus non-structural protein 3 contains a serine protease and an RNA helicase. Protease cleaves the genomeencoded polyprotein and inactivates cellular proteins required for innate immunity. Protease has emerged as an important target for the development of antiviral therapeutics, but drug resistance has turned out to be an obstacle in the clinic. Helicase is required for both genome replication and virus assembly. Mechanistic and structural studies of helicase have hurled this enzyme into a prominent position in the field of helicase enzymology. Nevertheless, studies of helicase as an antiviral target remain in their infancy.

Journal of Biological Chemistry, 2013

Background: The motif D loop in poliovirus RNA-dependent RNA polymerase is important for catalysi... more Background: The motif D loop in poliovirus RNA-dependent RNA polymerase is important for catalysis and fidelity. Results: A vaccine-derived mutation in motif D decreases RdRp fidelity by changing motif D conformational dynamics. Conclusion: Non-conserved residues of motif D can alter RdRp function. Significance: Motif D of the RdRp may be a universal target permitting creation of enzymes with perturbed fidelity and viruses with reduced virulence. . 3 The abbreviations used are: RdRp, RNA-dependent RNA polymerase; cPVR, poliovirus receptor; HSQC, heteronuclear single quantum correlation; IACUC, Institutional Animal Care and Use Committee; MD, molecular dynamics; PV, poliovirus; SDKIE, solvent deuterium kinetic isotope effect; sym/sub, symmetrical primer-template substrate; HRV16, human rhinovirus serotype 16.