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Ibrahim Moustafa

Assistant Research Professor of Biochemistry and Molecular Biology (Hafenstein Lab)
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The Hafenstein Lab

About Me

I am Ibrahim Moustafa, born in Cairo, Egypt.  During my early school years, I discovered my love for mathematics and science and dreamed to be a scientist.  Because of that, I chose to study Chemistry as my major at Alexandria University in Egypt. One thing that fascinated me as a chemistry student was the relationship between structure and function of molecules.  As a graduate student, I became interested in studying protein structures using X-ray crystallography and molecular modeling techniques. I obtained my Ph.D. degree in Structural Biology from St Andrews University, UK in 2004. I moved to USA with my family in 2004 to do my first postdoctoral training at UCSC. During my postdoctoral training, the interests in exploring the structures of these magnificent molecules continued to increase, and extended to go beyond the static view obtained from crystallography. In 2006, I joined Dr. Cameron’s lab at PSU as a Research Associate and stayed for a decade. In 2016, I accepted a teaching position at Bowdoin college, ME; in 2017 I accepted another teaching position at Beloit college, WI. In Sep 2018, I re-joined PSU to work with Dr. Hafenstein’s group as an Assistant Research Professor. Currently, my research is focused on elucidating structures of infectious viruses using Cryo-EM and structure-based drug design of new antivirals.


Research Interest

My research interest is to elucidate the structure-dynamics-function relationships of proteins in their native contexts. I use a combination of experimental and theoretical techniques including, Cryo-EM, X-ray crystallography, SAXS/WAXS, molecular modeling, docking and MD simulations to address fundamental biological questions. Currently, my research activity is mainly focused on structural biology of viruses and structure-based drug design of antivirals.


Research Summary

During my graduate study, I conducted work on Vibrio cholera neuraminidase (VCNA). Cholera is an ancient epidemic and pandemic worldwide disease; the pathogenesis of cholera involves several virulence factors secreted by the bacterium including the VCNA, which plays a significant role in the cholera disease.  VCNA is a part of the secreted mucinase complex, which cleaves the terminal sialic acid from the higher gangliosides to produce the GM1 ganglioside, the receptor of cholera toxin —the main virulence factor of cholera. By the time I started my Ph.D. work, the structural basis underlying the enzyme function was unknown.  In addition, the functional role of the two lectin-like domains flanking the central domain, which harbors the catalytic site of the neuraminidase, was a mystery. My crystallographic work uncovered the function of the N-terminal lectin-like domain that shown to specifically recognize sialic acid, promoting the attachment of the cholera enzyme to the surface of the hosting cells. Of note, the binding affinity of the N- lectin-like domain (classified as Carbohydrate Binding Domain 40, or CBM40) is one of the highest affinity reported for recognition of a monosaccharide by CBM.  This finding laid the ground to the development of a biological reagent (multivalent module of CBM) that have broad application in glycobiology. The solved structure of VCNA complexes with substrate and inhibitors combined with NMR study provided deep insights into the mechanism of VCNA. In that work I did all aspects of X-ray crystallography from protein expression and purification to data collection and structure determination. I also used NMR to characterize intermediates and ITC to do determine the binding affinity. 

Moustafa Research

During my 1st postdoctoral training at UCSC, I worked on flavoenzymes. L-amino acid oxidase (LAAO) is an FAD-dependent enzyme that catalyzes the oxidative deamination of L-amino acids to produce α-keto acids and hydrogen peroxide.  It is widely distributed in both prokaryotic and eukaryotic organisms; LAAO is major constituent of snake venom and thought to contribute to the toxicity of the venom.  LAAO is a dimeric glycosylated flavoenzyme, it exhibits apoptosis inducing effects as well as anti-bacterial and anti-HIV activities.  Trapping and structurally characterizing a true intermediate of LAAO in a defined oxidation state during the oxidative-half reaction was largely sought to gain mechanistic information for this important class of enzymes. The 1.8 Ǻ resolution structure of LAAO, from snake venom in complex with L-phenylalanine revealed the substrate bound to the reduced flavin, showed a dynamic active site and provided deeper insights into the mechanism of LAAO. In addition, inspection of the crystal structure revealed a Y-shaped channel system connecting the active site to the exterior of the enzyme.  The Y-channel proposed to provide two separate pathways: one was suggested for the entry of the O2 substrate, and the other was suggested for the release of H2O2 that is produced during the catalytic reaction.  Of interest, the channel would direct the H2O2 product to the exterior surface of the enzyme near the glycan moiety, thought to anchor the enzyme to the host cell.  This arrangement of the H2O2 channel provided a plausible explanation for the ability of the enzyme to localize H2O2 to the targeted cells and thus induce the apoptotic effect. Another flavoenzyme investigated to understand the structural basis of the mechanism is Cholestrol Oxidase, a bacterial flavoenzyme that catalyzes the first reaction in cholesterol metabolism. The Cholesterol Oxidase enzyme has many applications including its usage as a biological tool and in biocatalysis for the production of a number of steroids. We were able to determine the the sub-Angestom resolution (0.85 Ả) of the cholesterol oxidase under different conditions of pH, providing unprecedented details of the enzyme active site, and explained the reversible pH-dependent loss of oxidation activity of the enzyme. The very high resolution crystal structure at high pH unraveled a previously unknown mechanism for stabilization of the FAD cofactor. 

Moustafa Research

In Cameron’s lab at PSU, the primary focus of my research was to understand the factors controlling fidelity of RNA polymerases from picornaviruses. The virus-encoded RNA-dependent RNA polymerase (RdRp) is central to the multiplication of positive-strand RNA viruses. This enzyme, therefore, represents an attractive target for the development of antivirals. To obtain key insights into the enzyme mechanism I explored the dynamics of different RdRps to discover the connection between RdRp dynamics and incorporation fidelity. I was the leading scientist in this computational study. 

Moustafa Research

I also worked on a project to investigate the mitochondrial transcription machinery of mammalian cells. Mammalian mitochondria contain their own DNA genome (mtDNA) whose transcription is regulated by proteins encoded by nuclear genes that are imported to the mitochondria.  From previous studies, the components of the mitochondrial transcription machinery have been identified, including the bacteriophage-like RNA polymerase (mtRNAP or POLRMT), the mitochondrial transcription factor A (mtTFA or TFAM), and the mitochondrial transcription factor B2 (mtTFB2 or TFB2M).  In mammalian systems, TFB2M is indispensable for transcription initiation. Structural information for POLRMT and TFAM from humans is available; however, there is no available structure for TFB2M. In our study, three-dimensional structure of TFB2M from humans was modeled using a combination of homology modeling and small-angle X-ray scattering (SAXS). The TFB2M structural model added substantively to our understanding of TFB2M function. An explanation for the low or absent RNA methyltransferase activity was provided. A putative nucleic acid-binding site was revealed. The amino and carboxy termini, while likely lacking defined secondary structure, appeared to adopt compact, globular conformations, thus “capping” the ends of the protein. Finally, sites of interaction of TFB2M with other factors, protein and/or nucleic acid, were suggested by the identification of species-specific clusters on the surface of the protein. I was the leading scientist in this work, did the experimental and theoretical work.

In another study, we explored the structure-dynamics-function relationships of the multifunctional protein 3CD protease from poliovirus. 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 was unclear, since crystal structure of 3CD revealed “beads-on-a-string” structure with no significant structural differences compared to the fully processed proteins. I 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 I 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. I was the leading scientist in this project for both the experimental and theoretical parts. [Moustafa_image 4]

Moustafa Research

Currently, my research activity is mainly focused on applying cryo-EM to resolve structures of viruses and proteins at high resolutions & using the structure-based drug design approach to discover new antivirals.

My teaching experience is limited compared to my career in research. I taught introductory chemistry and biochemistry courses at Bowdoin College in Maine and Beloit College in Wisconsin. 


Selected Publications

  • Jingjing Shi, Jacob M. Perryman, Xiaorong Yang, Xinran Liu, Derek M. Musser, Alyson K. Boehr, Ibrahim M. Moustafa, Jamie J. Arnold, Craig E. Cameron and David D. Boehr. Rational control of viral RNA-dependent RNA polymerase fidelity by modulating motif D loop conformational dynamics. Biochemistry. 58(36): 3735-3743 (2019)
  • Dirar Homouz, Kwee Hong Joyce-Tan, Mohd Shahir Shamsir, Ibrahim M. Moustafa, and Haitham T. Idriss. Molecular dynamics simulations suggest changes in electrostatic interactions as a potential mechanism through which serine phosphorylation inhibits DNA polymerase β activity. Journal of Molecular Graphics and Modelling. 84: 236-241 (2018)
  • Djoshkun Shengjuler, Yan M. Chan, Simou Sun, Ibrahim M. Moustafa, ZhenLu Li, David W. Gohara, Matthias Buck, Paul S. Cremer, David D. Boehr and Craig E. Cameron. The RNA-binding site of poliovirus 3C protein doubles as a phosphoinositide-binding domain. Structure. 25: 1875-1886 (2017)
  • Xiaorong Yang, Xinran Liu, Derek M. Musser, Ibrahim M. Moustafa, Jamie J. Arnold, Craig E. Cameron and David D. Boehr. Triphosphate Re-orientation of the Incoming Nucleotide as a Fidelity Checkpoint in Viral RNA-dependent RNA polymerases. J. Biol. Chem. 292: 3810-3826 (2017)
  • Craig E. Cameron, Ibrahim M. Moustafa and Jamie J Arnold. Fidelity of nucleotide incorporation by the RNA-dependent RNA polymerase from poliovirus. In: Laurie S. Kaguni and Marcos T. Oliveira, editors, The Enzymes, Vol. 39, Burlington: Academic Press, 2016, pp.293-323
  • Yan M. Chan, Ibrahim M. Moustafa, Neela Yennawar, Jamie J. Arnold, Craig E. Cameron and David D. Boehr. Conformational adaptation in the multifunctional picornaviral 3C protease. Structure. 24: 509-517 (2016)
  • Ibrahim M. Moustafa, Akira Uchida, Neela Yennawar and Craig E. Cameron. Conformational ensemble of the poliovirus 3CD precursor obtained by MD simulations and confirmed by SAXS: A strategy to expand the viral proteome?. Viruses. 7: 5962-5986 (2015)
  • Ibrahim M. Moustafa, Akira Uchida, Neela Yennawar and Craig E. Cameron. Structural models of mammalian mitochondrial transcription factor B2. BBA Gene Regulatory Mechanisms. 1849: 987-1002 (2015)
  • Greta A. Van Slyke, Jamie J. Arnold, Sara B. Griesemer, Ibrahim M. Moustafa, Laura D. Kramer, Craig E. Cameron, and Alexander T. Ciota. Template-specific fidelity alterations associated with West Nile virus attenuation in mosquitoes. PLOS Pathogens. 11 (6): e1005009 (2015)
  • Ibrahim M. Moustafa, Victoria K. Korboukh, Jamie J. Arnold, Eric D. Smidansky, Laura L. Marcotte, David W. Gohara, Xiaorong Yang, Maria A. SanchezFarran, David Filman, Janna K. Maranas, David D. Boehr, James M. Hogle, Coray M. Colina and Craig E. Cameron. Structural Dynamics as a Contributor to Error-prone Replication by RNA-dependent RNA Polymerase. J. Biol. Chem. 289: 36229-36248 (2014)
  • David D. Boehr, Jamie J. Arnold, Ibrahim M. Moustafa and Craig E. Cameron. Structure, dynamics and fidelity of RNA-dependent RNA polymerases. In “Nucleic Acids and Molecular Biology” (pp. 309-333). Bujnicki, J. (Eds). Springer. (2014)
  • Xinran Liu, Xiaorong Yang, Cheri A. Lee, Ibrahim M. Moustafa, Eric D. Smidansky, David Lum, Jamie Arnold, Craig Cameron, and David D. Boehr. Vaccine-derived mutation in motifD of poliovirus RNA-dependent RNA polymerase lowers nucleotide incorporation fidelity. J. Biol. Chem. 288: 32753-32765 (2013)
  • Hujun Shen, Ibrahim M. Moustafa, Craig E. Cameron and Coray M. Colina..Exploring the dynamics of four RNA-dependent RNA polymerases by coarsegrained model. J. Phys. Chem. B 116: 14515-14524 (2012)
  • Jamie J. Arnold, Eric Smidansky, Ibrahim M. Moustafa, and Craig E. Cameron. Human mitochondrial RNA polymerase: Structure-Function, Mechanism and Inhibition. Biochem. Biophys. Acta 1819: 948-960 (2012)
  • Maria F. Lodeiro, Akira Uchida, Megan Bestwick, Ibrahim M. Moustafa, Jamie J. Arnold. Gerald S. Shadel and Craig E. Cameron. Transcription from the heavy-strand promoter of human mitochondrial DNA is repressed by transcription factor A in vitro. PNAS 109: 6513-6518 (2012)
  • Ibrahim M. Moustafa, Hujun Shen, Brandon Morton, Coray M. Colina, and Craig E. Cameron. Molecular dynamics simulations of viral RNA polymerases link conserved and correlated motions of functional elements to fidelity. J. Mol. Biol. 410: 159-181 (2011)
  • A. Anandan, C. Vallet, T. Cyole, Ibrahim M. Moustafa and Alice Vrielink..Crystallization and preliminary diffraction analysis of an engineered cephalosporin acylase. Acta Cryst. F66: 808-810 (2010)
  • Kevin D. Raney, Suresh D. Sharma, Ibrahim M. Moustafa and Craig E. Cameron. Hepatitis C virus non-structural protein 3 (HCV NS3): a multifunctional antiviral target. J. Biol. Chem. 285: 22725-22731 (2010)
  • Craig E. Cameron, Hyung Suk Oh and Ibrahim M. Moustafa. Expanding knowledge of P3 proteins in the poliovirus lifecycle. Future Microbiol. 5: 867-881 (2010)
  • Maria F. Lodeiro, Akira Uchida, Jamie J. Arnold, Shelley L. Reynolds, Ibrahim M. Moustafa and Craig E. Cameron. Identification of multiple rate-limiting steps during the human mitochondrial transcription cycle in vitro. J. Biol. Chem. 285: 16387-16402 (2010)
  • Craig E. Cameron, Ibrahim M. Moustafa, and Jamie J. Arnold. Dynamics: the missing link between structure and function of viral RNA-dependent RNA polymerase?. Currr. Opin. Struct. Biol. 19: 768-774 (2009)
  • Christian Castro, Eric D. Smidansky, Jamie J. Arnold, Kenneth R. Maksimchuk, Ibrahim M. Moustafa, Akira Uchida, Matthias Götte, William Konigsberg, and Craig E. Cameron. Nucleic acid polymerases use a general acid for nucleotidyl transfer. Nat. Struct. Mol. Biol. 16: 212-218 (2009)
  • Miaoking Shen, Zachary J. Reitman, Yan Zaho, Ibrahim M. Moustafa, Qixin Wang, Jamie J. Arnold, Harsh B. Pathak, and Craig E. Cameron. Picornavirus Genome Replication: Identification of the surface of poliovirus (PV) 3C dimer that interacts with 3Dpol during VPg uridylylation and construction of a structural model for the PV 3C2-3Dpol complex. J. Biol. Chem. 283: 875-888 (2008)
  • Amero C. D., Jamie J. Arnold, Ibrahim M. Moustafa, Craig E. Cameron, and M. P. Foster. Identification of the oriI-binding site of Poliovirus 3C protein by nuclear magnetic resonance spectroscopy. J. Virol. 82: 4363-4370 (2008)
  • Ibrahim M. Moustafa, Scott Foster, Artem Y. Lyubimov, and Alice Vrielink. Crystal structure of LAAO from Calloselasema rhodostoma with L-Phenylalanine substrate: Insights into structure and mechanism. J. Mol. Biol. 364: 991-1002 (2006)
  • Artem Y. Lyubimov, Paula I. Lario, Ibrahim M. Moustafa, and Alice Vrielink. Atomic resolution crystallography reveals how changes in pH shape the microenvironment of a protein. Nat. Chem. Biol. 2: 259-264 (2006)
  • Ibrahim M. Moustafa, Helen Connaris, Margaret Taylor, Viateslav Zaitsev, Jennifer C. Wilson, Milton J. Kiefel, Mark von Itzstein, and Garry Taylor. Sialic acid recognition by Vibro cholerae neuraminidase. J. Biol. Chem. 279: 40819-40826 (2004)
  • Helen Connaris, Toru Takimoto, Rupert Russell, Susan Crennell, Ibrahim M. Moustafa, Allen Portner, and Garry Taylor. Probing sialic acid binding site of the Haemagglutinin- Neuraminidase of Newcastle Disease Virus: Identification of key amino acids involved in cell binding, catalysis, and fusion. J. Virol. 76: 1816-1824 (2002)