28 results on '"Celia A. Schiffer"'
Search Results
2. Interactions of APOBEC3s with DNA and RNA
- Author
-
Hiroshi Matsuo, Shurong Hou, Atanu Maiti, and Celia A. Schiffer
- Subjects
chemistry.chemical_classification ,0303 health sciences ,Innate immune system ,viruses ,Deamination ,RNA ,DNA ,biochemical phenomena, metabolism, and nutrition ,Article ,Epitope ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Enzyme ,chemistry ,Biochemistry ,Structural Biology ,Nucleic acid ,APOBEC Deaminases ,Molecular Biology ,030217 neurology & neurosurgery ,Cytosine ,030304 developmental biology - Abstract
APOBEC3 enzymes are key enzymes in our innate immune system regulating antiviral response in HIV and unfortunately adding diversity in cancer as they deaminate cytosine. Seven unique single and double domain APOBEC3s provide them with unique activity and specificity profiles for this deamination. Recent crystal and NMR structures of APOBEC3 complexes are unraveling the variety of epitopes involved in binding nucleic acids, including at the catalytic site, elsewhere on the catalytic domain and in the inactive N-terminal domain. The interplay between these diverse interactions is critical to uncovering the mechanisms by which APOBEC3s recognize and process their substrates.
- Published
- 2021
3. Non-canonical pattern recognition of a pathogen-derived metabolite by a nuclear hormone receptor identifies virulent bacteria in C. elegans
- Author
-
Nicholas D. Peterson, Samantha Y. Tse, Qiuyu Judy Huang, Khursheed A. Wani, Celia A. Schiffer, and Read Pukkila-Worley
- Subjects
Infectious Diseases ,Immunology ,Immunology and Allergy - Published
- 2023
4. HIV-1 Protease Uses Bi-Specific S2/S2′ Subsites to Optimize Cleavage of Two Classes of Target Sites
- Author
-
Charles W. Carter, Ean Spielvogel, Marc Potempa, Celia A. Schiffer, Ronald Swanstrom, Amy Rogers, Nese Kurt Yilmaz, Ellen A. Nalivaika, and Sook Kyung Lee
- Subjects
Models, Molecular ,0301 basic medicine ,Steric effects ,Conformational change ,Proline ,Protein Conformation ,Stereochemistry ,Cleavage (embryo) ,Article ,Substrate Specificity ,03 medical and health sciences ,Scissile bond ,HIV Protease ,HIV-1 protease ,Structural Biology ,Side chain ,Amino Acids ,Molecular Biology ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,Binding Sites ,030102 biochemistry & molecular biology ,biology ,030302 biochemistry & molecular biology ,3. Good health ,Amino acid ,Kinetics ,030104 developmental biology ,chemistry ,Proteolysis ,HIV-1 ,biology.protein ,Hydrophobic and Hydrophilic Interactions - Abstract
Retroviral proteases (PR) have a unique specificity that allows cleavage of sites with or without a P1’ proline. A P1’ proline is required at the MA/CA cleavage site due to its role in a post-cleavage conformational change in the capsid protein. However, the HIV-1 PR prefers to have large hydrophobic amino acids flanking the scissile bond, suggesting PR recognizes two different classes of substrate sequences. We analyzed the cleavage rate of over 150 iterations of six different HIV-1 cleavage sites to explore rate determinants of cleavage. We found that cleavage rates are strongly influenced by the two amino acids flanking the amino acids at the scissile bond (P2-P1/P1’-P2’), with two complementary sets of rules. When P1’ is proline, the P2 side chain interacts with a polar region in the S2 subsite of the PR, while the P2’ amino acid interacts with a hydrophobic region of the S2’ subsite. When P1’ is not proline, the orientations of the P2 and P2’ side chains with respect to the scissile bond are reversed; P2 residues interact with a hydrophobic face of the S2 subsite while the P2’ amino acid usually engages hydrophilic amino acids in the S2’ subsite. These results reveal that the HIV-1 PR has evolved bi-functional S2 and S2’ subsites to accommodate the steric effects imposed by a P1’ proline on the orientation of P2 and P2’ substrate side chains. These results also suggest a new strategy for inhibitor design to engage the multiple specificities in these subsites.
- Published
- 2018
5. Structural basis of substrate specificity in human cytidine deaminase family APOBEC3s
- Author
-
Shurong Hou, Jeong Min Lee, Nese Kurt Yilmaz, Wazo Myint, Hiroshi Matsuo, and Celia A. Schiffer
- Subjects
Models, Molecular ,0301 basic medicine ,pMD, parallel molecular dynamics ,Molecular model ,ASBMB Award Article ,Deamination ,DNA, Single-Stranded ,specificity ,Crystallography, X-Ray ,Biochemistry ,Substrate Specificity ,03 medical and health sciences ,Catalytic Domain ,Neoplasms ,Humans ,Nucleotide ,APOBEC Deaminases ,Amino Acid Sequence ,structural analysis ,NTD, N-terminal domain ,ssDNA, single-strand DNA ,Molecular Biology ,chemistry.chemical_classification ,030102 biochemistry & molecular biology ,biology ,molecular modeling ,Chemistry ,C-terminus ,Nucleic acid sequence ,Active site ,Substrate (chemistry) ,APOBEC3 ,Cell Biology ,Cytidine deaminase ,CBE, cytosine base editor ,CTD, C-terminal domain ,molecular dynamics simulation ,030104 developmental biology ,Mutation ,biology.protein ,Protein Binding - Abstract
The human cytidine deaminase family of APOBEC3s (A3s) plays critical roles in both innate immunity and the development of cancers. A3s comprise seven functionally overlapping but distinct members that can be exploited as nucleotide base editors for treating genetic diseases. Although overall structurally similar, A3s have vastly varying deamination activity and substrate preferences. Recent crystal structures of ssDNA-bound A3s together with experimental studies have provided some insights into distinct substrate specificities among the family members. However, the molecular interactions responsible for their distinct biological functions and how structure regulates substrate specificity are not clear. In this study, we identified the structural basis of substrate specificities in three catalytically active A3 domains whose crystal structures have been previously characterized: A3A, A3B- CTD, and A3G-CTD. Through molecular modeling and dynamic simulations, we found an interdependency between ssDNA substrate binding conformation and nucleotide sequence specificity. In addition to the U-shaped conformation seen in the crystal structure with the CTC0 motif, A3A can accommodate the CCC0 motif when ssDNA is in a more linear (L) conformation. A3B can also bind both U- and L-shaped ssDNA, unlike A3G, which can stably recognize only linear ssDNA. These varied conformations are stabilized by sequence-specific interactions with active site loops 1 and 7, which are highly variable among A3s. Our results explain the molecular basis of previously observed substrate specificities in A3s and have implications for designing A3-specific inhibitors for cancer therapy as well as engineering base-editing systems for gene therapy.
- Published
- 2021
6. Cryo-EM structure of CtBP2 confirms tetrameric architecture
- Author
-
Steven R. Grossman, Dipankar Bandyopadhyay, Anne M. Jecrois, William E. Royer, M. Michael Dcona, Xiaoyan Deng, and Celia A. Schiffer
- Subjects
Mutant ,DNA-binding protein ,Article ,03 medical and health sciences ,CTBP1 ,Tetramer ,Cell Movement ,Structural Biology ,Catalytic Domain ,Transcriptional regulation ,Native state ,Humans ,T-Lymphoma Invasion and Metastasis-inducing Protein 1 ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,Chemistry ,Cryoelectron Microscopy ,030302 biochemistry & molecular biology ,Cell migration ,Cadherins ,HCT116 Cells ,CTBP2 ,Cell biology ,DNA-Binding Proteins ,Gene Expression Regulation, Neoplastic ,Alcohol Oxidoreductases ,Mutation ,Protein Multimerization ,Co-Repressor Proteins ,NADP ,Protein Binding - Abstract
C-terminal binding proteins 1 and 2 (CtBP1 and CtBP2) are transcriptional regulators that activate or repress many genes involved in cellular development, apoptosis, and metastasis. NADH-dependent CtBP activation has been implicated in multiple types of cancer and poor patient prognosis. Central to understanding activation of CtBP in oncogenesis is uncovering how NADH triggers protein assembly, what level of assembly occurs, and if oncogenic activity depends upon such assembly. Here, we present the cryoelectron microscopic structures of two different constructs of CtBP2 corroborating that the native state of CtBP2 in the presence of NADH is tetrameric. The physiological relevance of the observed tetramer was demonstrated in cell culture, showing that CtBP tetramer-destabilizing mutants are defective for cell migration, transcriptional repression of E-cadherin, and activation of TIAM1. Together with our cryoelectron microscopy studies, these results highlight the tetramer as the functional oligomeric form of CtBP2.
- Published
- 2021
7. NAD(H) phosphates mediate tetramer assembly of human C-terminal binding protein (CtBP)
- Author
-
William E. Royer, Celia A. Schiffer, and Jeffry C. Nichols
- Subjects
Models, Molecular ,0301 basic medicine ,Dehydrogenase ,Transcription coregulator ,Biochemistry ,DNA-binding protein ,MALS ,03 medical and health sciences ,Tetramer ,tetrameric assembly ,TCEP, tris(2-carboxyethyl) phosphine ,CtBP, C-terminal binding protein ,medicine ,Humans ,structural biology ,Nucleotide ,CtBP ,crystallography ,Molecular Biology ,chemistry.chemical_classification ,030102 biochemistry & molecular biology ,Chemistry ,NAD(H) ,MALS, multiangle light scattering ,D2-HDH, D-isomer-specific 2-hydroxyacid dehydrogenase ,Cell Biology ,NAD ,Adenosine ,DNA-Binding Proteins ,Alcohol Oxidoreductases ,transcription coregulator ,SEC, size-exclusion column ,030104 developmental biology ,Structural biology ,dehydrogenase ,Biophysics ,cancer target ,NAD+ kinase ,Protein Multimerization ,Co-Repressor Proteins ,NADP ,Research Article ,medicine.drug - Abstract
C-terminal binding proteins (CtBPs) are cotranscriptional factors that play key roles in cell fate. We have previously shown that NAD(H) promotes the assembly of similar tetramers from either human CtBP1 and CtBP2 and that CtBP2 tetramer destabilizing mutants are defective for oncogenic activity. To assist structure-based design efforts for compounds that disrupt CtBP tetramerization, it is essential to understand how NAD(H) triggers tetramer assembly. Here, we investigate the moieties within NAD(H) that are responsible for triggering tetramer formation. Using multiangle light scattering (MALS), we show that ADP is able to promote tetramer formation of both CtBP1 and CtBP2, whereas AMP promotes tetramer assembly of CtBP1, but not CtBP2. Other NAD(H) moieties that lack the adenosine phosphate, including adenosine and those incorporating nicotinamide, all fail to promote tetramer assembly. Our crystal structures of CtBP1 with AMP reveal participation of the adenosine phosphate in the tetrameric interface, pinpointing its central role in NAD(H)-linked assembly. CtBP1 and CtBP2 have overlapping but unique roles, suggesting that a detailed understanding of their unique structural properties might have utility in the design of paralog-specific inhibitors. We investigated the different responses to AMP through a series of site-directed mutants at 13 positions. These mutations reveal a central role for a hinge segment, which we term the 120s hinge that connects the substrate with coenzyme-binding domains and influences nucleotide binding and tetramer assembly. Our results provide insight into suitable pockets to explore in structure-based drug design to interfere with cotranscriptional activity of CtBP in cancer.
- Published
- 2021
8. Improving Viral Protease Inhibitors to Counter Drug Resistance
- Author
-
Celia A. Schiffer, Ronald Swanstrom, and Nese Kurt Yilmaz
- Subjects
0301 basic medicine ,Microbiology (medical) ,Drug ,media_common.quotation_subject ,medicine.medical_treatment ,Hepatitis C virus ,030106 microbiology ,HIV Infections ,Drug resistance ,Computational biology ,medicine.disease_cause ,Microbiology ,Article ,03 medical and health sciences ,HIV Protease ,HIV-1 protease ,Virology ,Drug Resistance, Viral ,medicine ,Humans ,Protease Inhibitors ,media_common ,Protease ,biology ,Rational design ,Hepatitis C ,On resistance ,030104 developmental biology ,Infectious Diseases ,Viral protease ,Drug Design ,biology.protein - Abstract
Drug resistance is a major problem in health care, undermining therapy outcomes and necessitating novel approaches to drug design. Extensive studies on resistance to viral protease inhibitors, particularly those of HIV-1 and hepatitis C virus (HCV) protease, revealed a plethora of information on the structural and molecular mechanisms underlying resistance. These insights led to several strategies to improve viral protease inhibitors to counter resistance, such as exploiting the essential biological function and leveraging evolutionary constraints. Incorporation of these strategies into structure-based drug design can minimize vulnerability to resistance, not only for viral proteases but for other quickly evolving drug targets as well, toward designing inhibitors one step ahead of evolution to counter resistance with more intelligent and rational design.
- Published
- 2016
9. Illuminating the Structural Determinants for Tetrameric Assembly of Oncogenic CtBP to Guide Inhibitor Design
- Author
-
Celia A. Schiffer, Jeffry C. Nichols, and William E. Royer
- Subjects
Biophysics - Published
- 2020
10. A Direct Interaction with RNA Dramatically Enhances the Catalytic Activity of the HIV-1 Protease In Vitro
- Author
-
Celia A. Schiffer, Ellen A. Nalivaika, Sook-Kyung Lee, Ronald Swanstrom, Debra A. Ragland, and Marc Potempa
- Subjects
Models, Molecular ,Protein Conformation ,medicine.medical_treatment ,Proteolysis ,Allosteric regulation ,DNA, Single-Stranded ,In Vitro Techniques ,Cleavage (embryo) ,Article ,Catalysis ,HIV Protease ,RNA, Transfer ,HIV-1 protease ,Structural Biology ,medicine ,Humans ,Molecular Biology ,chemistry.chemical_classification ,Protease ,biology ,medicine.diagnostic_test ,Virus Assembly ,Virion ,RNA ,Cell biology ,Enzyme ,chemistry ,Biochemistry ,biology.protein ,Nucleic acid ,Protein Multimerization ,Allosteric Site ,Protein Binding - Abstract
Though the steps of human immunodeficiency virus type 1 (HIV-1) virion maturation are well documented, the mechanisms regulating the proteolysis of the Gag and Gag-Pro-Pol polyproteins by the HIV-1 protease (PR) remain obscure. One proposed mechanism argues that the maturation intermediate p15NC must interact with RNA for efficient cleavage by the PR. We investigated this phenomenon and found that processing of multiple substrates by the HIV-1 PR was enhanced in the presence of RNA. The acceleration of proteolysis occurred independently from the substrate's ability to interact with nucleic acid, indicating that a direct interaction between substrate and RNA is not necessary for enhancement. Gel-shift assays demonstrated the HIV-1 PR is capable of interacting with nucleic acids, suggesting that RNA accelerates processing reactions by interacting with the PR rather than the substrate. All HIV-1 PRs examined have this ability; however, the HIV-2 PR does not interact with RNA and does not exhibit enhanced catalytic activity in the presence of RNA. No specific sequence or structure was required in the RNA for a productive interaction with the HIV-1 PR, which appears to be principally, though not exclusively, driven by electrostatic forces. For a peptide substrate, RNA increased the kinetic efficiency of the HIV-1 PR by an order of magnitude, affecting both turnover rate ( k cat ) and substrate affinity ( K m ). These results suggest that an allosteric binding site exists on the HIV-1 PR and that HIV-1 PR activity during maturation could be regulated in part by the juxtaposition of the enzyme with virion-packaged RNA.
- Published
- 2015
11. Tetrameric Assembly of the Oncogenic C-Terminal Binding Proteins
- Author
-
M. Michael Dcona, Brendan J. Hilbert, William E. Royer, Anne M. Jecrois, Steven R. Grossman, Andrew G. Bellesis, and Celia A. Schiffer
- Subjects
Stereochemistry ,Chemistry ,Biophysics ,C-terminal binding - Published
- 2019
12. Substrate Envelope-Designed Potent HIV-1 Protease Inhibitors to Avoid Drug Resistance
- Author
-
Akbar Ali, Madhavi N. L. Nalam, G. S. Kiran Kumar Reddy, Hong Cao, Michael D. Altman, Celia A. Schiffer, Tariq M. Rana, Nese Kurt Yilmaz, Saima Ghafoor Anjum, Bruce Tidor, Massachusetts Institute of Technology. Department of Biological Engineering, Massachusetts Institute of Technology. Department of Electrical Engineering and Computer Science, Altman, Michael D., and Tidor, Bruce
- Subjects
Drug ,media_common.quotation_subject ,medicine.medical_treatment ,Static Electricity ,Clinical Biochemistry ,Drug resistance ,Plasma protein binding ,010402 general chemistry ,medicine.disease_cause ,01 natural sciences ,Biochemistry ,Article ,Substrate Specificity ,03 medical and health sciences ,HIV Protease ,HIV-1 protease ,Microsomes ,Drug Resistance, Viral ,Drug Discovery ,medicine ,Humans ,HIV Protease Inhibitor ,Molecular Biology ,030304 developmental biology ,media_common ,Pharmacology ,0303 health sciences ,Mutation ,Protease ,biology ,HIV Protease Inhibitors ,General Medicine ,Virology ,3. Good health ,0104 chemical sciences ,Multiple drug resistance ,Kinetics ,Drug Design ,HIV-1 ,biology.protein ,Molecular Medicine ,Protein Binding - Abstract
The rapid evolution of HIV under selective drug pressure has led to multidrug resistant (MDR) strains that evade standard therapies. We designed highly potent HIV-1 protease inhibitors (PIs) using the substrate envelope model, which confines inhibitors within the consensus volume of natural substrates, providing inhibitors less susceptible to resistance because a mutation affecting such inhibitors will simultaneously affect viral substrate processing. The designed PIs share a common chemical scaffold but utilize various moieties that optimally fill the substrate envelope, as confirmed by crystal structures. The designed PIs retain robust binding to MDR protease variants and display exceptional antiviral potencies against different clades of HIV as well as a panel of 12 drug-resistant viral strains. The substrate envelope model proves to be a powerful strategy to develop potent and robust inhibitors that avoid drug resistance., National Institute of General Medical Sciences (U.S.) (Grant P01-GM66524), National Institute of General Medical Sciences (U.S.) (Grant AI41404), National Institute of General Medical Sciences (U.S.) (Grant AI43198), National Institute of General Medical Sciences (U.S.) (Grant GM065418), National Institute of General Medical Sciences (U.S.) (Grant GM082209), United States. American Recovery and Reinvestment Act of 2009 (Supplement P01GM066524-08S1)
- Published
- 2013
13. Crystal Structure of the DNA Cytosine Deaminase APOBEC3F: The Catalytically Active and HIV-1 Vif-Binding Domain
- Author
-
Hiroshi Matsuo, Michael A. Carpenter, Shivender M.D. Shandilya, Rebecca M. McDougle, Mohan Somasundaran, Brett D. Anderson, Celia A. Schiffer, Takahide Kouno, Anurag Rathore, Reuben S. Harris, John S. Albin, Markus-Frederik Bohn, Ahkillah N. Davis, Leah Evans, JingYing Zhang, and Yongjian Lu
- Subjects
Models, Molecular ,Protein Conformation ,viruses ,Crystal structure ,Biology ,Crystallography, X-Ray ,Article ,Catalysis ,Cytosine Deaminase ,03 medical and health sciences ,chemistry.chemical_compound ,Protein structure ,Structural Biology ,Hydrolase ,vif Gene Products, Human Immunodeficiency Virus ,Structural motif ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,030302 biochemistry & molecular biology ,Cytosine deaminase ,virus diseases ,biochemical phenomena, metabolism, and nutrition ,3. Good health ,chemistry ,Biochemistry ,HIV-1 ,Sequence motif ,DNA ,Binding domain - Abstract
SummaryHuman APOBEC3F is an antiretroviral single-strand DNA cytosine deaminase, susceptible to degradation by the HIV-1 protein Vif. In this study the crystal structure of the HIV Vif binding, catalytically active, C-terminal domain of APOBEC3F (A3F-CTD) was determined. The A3F-CTD shares structural motifs with portions of APOBEC3G-CTD, APOBEC3C, and APOBEC2. Residues identified to be critical for Vif-dependent degradation of APOBEC3F all fit within a predominantly negatively charged contiguous region on the surface of A3F-CTD. Specific sequence motifs, previously shown to play a role in Vif susceptibility and virion encapsidation, are conserved across APOBEC3s and between APOBEC3s and HIV-1 Vif. In this structure these motifs pack against each other at intermolecular interfaces, providing potential insights both into APOBEC3 oligomerization and Vif interactions.
- Published
- 2013
- Full Text
- View/download PDF
14. Context Surrounding Processing Sites Is Crucial in Determining Cleavage Rate of a Subset of Processing Sites in HIV-1 Gag and Gag-Pro-Pol Polyprotein Precursors by Viral Protease
- Author
-
Celia A. Schiffer, Marc Potempa, Ayşegül Özen, Madhavi Kolli, Ronald Swanstrom, and Sook-Kyung Lee
- Subjects
Proteases ,Polyproteins ,Viral protein ,viruses ,medicine.medical_treatment ,medicine.disease_cause ,Cleavage (embryo) ,gag Gene Products, Human Immunodeficiency Virus ,Biochemistry ,Substrate Specificity ,Scissile bond ,HIV Protease ,medicine ,Amino Acid Sequence ,Molecular Biology ,Peptide sequence ,HIV Long Terminal Repeat ,Protease ,biology ,Virus Assembly ,Virion ,Active site ,Cell Biology ,Molecular biology ,pol Gene Products, Human Immunodeficiency Virus ,Enzymology ,HIV-1 ,biology.protein - Abstract
Processing of the human immunodeficiency virus type 1 (HIV-1) Gag and Gag-Pro-Pol polyproteins by the HIV-1 protease (PR) is essential for the production of infectious particles. However, the determinants governing the rates of processing of these substrates are not clearly understood. We studied the effect of substrate context on processing by utilizing a novel protease assay in which a substrate containing HIV-1 matrix (MA) and the N-terminal domain of capsid (CA) is labeled with a FlAsH (fluorescein arsenical hairpin) reagent. When the seven cleavage sites within the Gag and Gag-Pro-Pol polyproteins were placed at the MA/CA site, the rates of cleavage changed dramatically compared with that of the cognate sites in the natural context reported previously. The rate of processing was affected the most for three sites: CA/spacer peptide 1 (SP1) (≈10-fold increase), SP1/nucleocapsid (NC) (≈10-30-fold decrease), and SP2/p6 (≈30-fold decrease). One of two multidrug-resistant (MDR) PR variants altered the pattern of processing rates significantly. Cleavage sites within the Pro-Pol region were cleaved in a context-independent manner, suggesting for these sites that the sequence itself was the determinant of rate. In addition, a chimera consisting of SP1/NC P4-P1 and MA/CA P1'-P4' residues (ATIM↓PIVQ) abolished processing by wild type and MDR proteases, and the reciprocal chimera consisting of MA/CA P4-P1 and SP1/NC P1'-4' (SQNY↓IQKG) was cleaved only by one of the MDR proteases. These results suggest that complex substrate interactions both beyond the active site of the enzyme and across the scissile bond contribute to defining the rate of processing by the HIV-1 PR.
- Published
- 2012
15. Molecular Mechanism of Resistance in a Clinically Significant Double-Mutant Variant of HCV NS3/4A Protease
- Author
-
Christos J. Petropoulos, N. KurtYilmaz, Florian Leidner, Wei Huang, Ashley N. Matthew, Celia A. Schiffer, Akbar Ali, and Alicia Newton
- Subjects
Cyclopropanes ,Models, Molecular ,0301 basic medicine ,Protein Conformation ,medicine.medical_treatment ,Hepatitis C virus ,Hepacivirus ,Drug resistance ,Molecular Dynamics Simulation ,Viral Nonstructural Proteins ,Crystallography, X-Ray ,medicine.disease_cause ,Antiviral Agents ,Article ,03 medical and health sciences ,Drug Resistance, Multiple, Viral ,Structural Biology ,Catalytic Domain ,Quinoxalines ,Catalytic triad ,medicine ,Humans ,Protease Inhibitors ,Protease inhibitor (pharmacology) ,Molecular Biology ,Sulfonamides ,Protease ,Chemistry ,Danoprevir ,Amides ,Virology ,030104 developmental biology ,Amino Acid Substitution ,Grazoprevir ,Paritaprevir ,Mutation ,Carbamates - Abstract
Despite significant progress in hepatitis C virus (HCV) protease inhibitor (PI) drug design, resistance remains a problem causing treatment failure. Double-substitution variants, notably Y56H/D168A, have emerged in patients who fail therapy with a PI-containing regimen. The resistance conferred by Asp168 substitutions has been well characterized and avoided in newer inhibitors. However, an additional mutation at Tyr56 confers resistance to even the most robust inhibitors. Here, we elucidate the molecular mechanisms of resistance for the Y56H/D168A variant against grazoprevir (and four analogs), paritaprevir, and danoprevir through inhibition assays, co-crystal structures, and molecular dynamics simulations. The PIs' susceptibility to Y56H/D168A varies, with those stacking on the catalytic His57 losing the most potency. For such inhibitors, the Y56H substitution disrupts favorable stacking interactions with the neighboring catalytic His57. This indirect mechanism of resistance threatens to cause multi-PI failure as all HCV PIs in clinical development rely on interactions with the catalytic triad.
- Published
- 2018
16. Rationale for More Diverse Inhibitors in Competition with Substrates in HIV-1 Protease
- Author
-
Nevra Özer, Celia A. Schiffer, and Turkan Haliloglu
- Subjects
Models, Molecular ,Anisotropic Network Model ,Stereochemistry ,Inhibitor complex ,Movement ,medicine.medical_treatment ,Biophysics ,Crystallography, X-Ray ,Ligands ,Binding, Competitive ,01 natural sciences ,03 medical and health sciences ,HIV Protease ,HIV-1 protease ,Catalytic Domain ,Drug Resistance, Viral ,0103 physical sciences ,medicine ,HIV Protease Inhibitor ,Binding site ,Protein Structure, Quaternary ,030304 developmental biology ,0303 health sciences ,Protease ,010304 chemical physics ,biology ,Protein ,Substrate (chemistry) ,Active site ,HIV Protease Inhibitors ,Kinetics ,HIV-1 ,biology.protein ,Anisotropy ,Protein Multimerization - Abstract
The structural fluctuations of HIV-1 protease in interaction with its substrates versus inhibitors were analyzed using the anisotropic network model. The directions of fluctuations in the most cooperative functional modes differ mainly around the dynamically key regions, i.e., the hinge axes, which appear to be more flexible in substrate complexes. The flexibility of HIV-1 protease is likely optimized for the substrates' turnover, resulting in substrate complexes being dynamic. In contrast, in an inhibitor complex, the inhibitor should bind and lock down to inactivate the active site. Protease and ligands are not independent. Substrates are also more flexible than inhibitors and have the potential to meet the dynamic distributions that are inherent in the protease. This may suggest a rationale and guidelines for designing inhibitors that can better fit the ensemble of binding sites that are dynamically accessible to the protease.
- Published
- 2010
17. Competition between Ski and CREB-binding Protein for Binding to Smad Proteins in Transforming Growth Factor-β Signaling
- Author
-
Hema Srinath, Celia A. Schiffer, Weijun Chen, Suvana S. Lam, William E. Royer, and Kai Lin
- Subjects
Models, Molecular ,animal structures ,Pentamer ,Smad Proteins ,SMAD ,Random hexamer ,Crystallography, X-Ray ,Binding, Competitive ,Biochemistry ,Transforming Growth Factor beta ,Proto-Oncogene Proteins ,Humans ,Protein activity ,CREB-binding protein ,Protein Structure, Quaternary ,Molecular Biology ,biology ,Isothermal titration calorimetry ,Cell Biology ,CREB-Binding Protein ,In vitro ,Cell biology ,DNA-Binding Proteins ,biology.protein ,human activities ,Protein Binding ,Signal Transduction ,Transforming growth factor - Abstract
The family of Smad proteins mediates transforming growth factor-beta (TGF-beta) signaling in cell growth and differentiation. Smads repress or activate TGF-beta signaling by interacting with corepressors (e.g. Ski) or coactivators (e.g. CREB-binding protein (CBP)), respectively. Specifically, Ski has been shown to interfere with the interaction between Smad3 and CBP. However, it is unclear whether Ski competes with CBP for binding to Smads and whether they can interact with Smad3 at the same binding surface on Smad3. We investigated the interactions among purified constructs of Smad, Ski, and CBP in vitro by size-exclusion chromatography, isothermal titration calorimetry, and mutational studies. Here, we show that Ski-(16-192) interacted directly with a homotrimer of receptor-regulated Smad protein (R-Smad), e.g. Smad2 or Smad3, to form a hexamer; Ski-(16-192) interacted with an R-Smad.Smad4 heterotrimer to form a pentamer. CBP-(1941-1992) was also found to interact directly with an R-Smad homotrimer to form a hexamer and with an R-Smad.Smad4 heterotrimer to form a pentamer. Moreover, these domains of Ski and CBP competed with each other for binding to Smad3. Our mutational studies revealed that domains of Ski and CBP interacted with Smad3 at a portion of the binding surface of the Smad anchor for receptor activation. Our results suggest that Ski negatively regulates TGF-beta signaling by replacing CBP in R-Smad complexes. Our working model suggests that Smad protein activity is delicately balanced by Ski and CBP in the TGF-beta pathway.
- Published
- 2007
18. Hydrophobic Sliding: A Possible Mechanism for Drug Resistance in Human Immunodeficiency Virus Type 1 Protease
- Author
-
Jennifer E. Foulkes-Murzycki, Celia A. Schiffer, and Walter R. P. Scott
- Subjects
PROTEINS ,Protein Conformation ,medicine.medical_treatment ,HUMDISEASE ,010402 general chemistry ,medicine.disease_cause ,01 natural sciences ,Article ,03 medical and health sciences ,Molecular dynamics ,Protein structure ,HIV Protease ,Structural Biology ,Drug Resistance, Viral ,medicine ,HIV Protease Inhibitor ,Amino Acids ,Molecular Biology ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,Mutation ,Protease ,biology ,Active site ,Hydrogen Bonding ,HIV Protease Inhibitors ,Protease inhibitor (biology) ,3. Good health ,0104 chemical sciences ,Amino acid ,Biochemistry ,chemistry ,biology.protein ,Hydrophobic and Hydrophilic Interactions ,medicine.drug - Abstract
SummaryHydrophobic residues outside the active site of HIV-1 protease frequently mutate in patients undergoing protease inhibitor therapy; however, the mechanism by which these mutations confer drug resistance is not understood. From analysis of molecular dynamics simulations, 19 core hydrophobic residues appear to facilitate the conformational changes that occur in HIV-1 protease. The hydrophobic core residues slide by each other, exchanging one hydrophobic van der Waal contact for another, with little energy penalty, while maintaining many structurally important hydrogen bonds. Such hydrophobic sliding may represent a general mechanism by which proteins undergo conformational changes. Mutation of these residues in HIV-1 protease would alter the packing of the hydrophobic core, affecting the conformational flexibility of the protease. Therefore these residues impact the dynamic balance between processing substrates and binding inhibitors, and thus contribute to drug resistance.
- Published
- 2007
- Full Text
- View/download PDF
19. Combating Susceptibility to Drug ResistanceLessons from HIV-1 Protease
- Author
-
Moses Prabu-Jeyabalan, Ellen A. Nalivaika, Nancy M. King, and Celia A. Schiffer
- Subjects
Pharmacology ,Drug ,biology ,media_common.quotation_subject ,Clinical Biochemistry ,General Medicine ,Biochemistry ,Virology ,HIV-1 protease ,Drug Discovery ,biology.protein ,Molecular Medicine ,Molecular Biology ,media_common - Published
- 2004
20. Combating Susceptibility to Drug Resistance
- Author
-
Moses Prabu-Jeyabalan, Celia A. Schiffer, Ellen A. Nalivaika, and Nancy M. King
- Subjects
Drug ,Modern medicine ,media_common.quotation_subject ,medicine.medical_treatment ,Clinical Biochemistry ,Computational biology ,Drug resistance ,Pharmacology ,010402 general chemistry ,01 natural sciences ,Biochemistry ,03 medical and health sciences ,HIV-1 protease ,Drug Discovery ,medicine ,Molecular Biology ,030304 developmental biology ,media_common ,0303 health sciences ,Protease ,biology ,Active site ,General Medicine ,3. Good health ,0104 chemical sciences ,Drug development ,biology.protein ,Molecular Medicine ,Function (biology) - Abstract
Drug resistance is a major obstacle in modern medicine. However, resistance is rarely considered in drug development and may inadvertently be facilitated, as many designed inhibitors contact residues that can mutate to confer resistance, without significantly impairing function. Contemporary drug design often ignores the detailed atomic basis for function and primarily focuses on disrupting the target's activity, which is necessary but not sufficient for developing a robust drug. In this study, we examine the impact of drug-resistant mutations in HIV-1 protease on substrate recognition and demonstrate that most primary active site mutations do not extensively contact substrates, but are critical to inhibitor binding. We propose a general, structure-based strategy to reduce the probability of drug resistance by designing inhibitors that interact only with those residues that are essential for function.
- Published
- 2004
- Full Text
- View/download PDF
21. Remembering Professor Amy Christine Anderson
- Author
-
Celia A. Schiffer
- Subjects
0301 basic medicine ,Pharmacology ,010405 organic chemistry ,Clinical Biochemistry ,Scientific thought ,Art history ,Environmental ethics ,Biology ,01 natural sciences ,Biochemistry ,0104 chemical sciences ,03 medical and health sciences ,030104 developmental biology ,Drug Discovery ,Molecular Medicine ,Molecular Biology ,Resistance (creativity) - Abstract
This summer we lost a scientific thought leader, brilliant colleague and researcher, and mentor and friend way before her time. Amy Anderson (1969 – 2016) realized that the Achilles’ heel of many microbial targets, particularly quickly evolving ones with a high propensity of resistance, was the enzyme dihydrofolate reductase (DHFR). Having received a BA in life sciences at MIT (1991) and PhD in biophysics (1997) at Harvard, Amy went on to UCSF to work with Robert M. Stroud, where she launched her career focus on small molecule inhibitors of the folate pathway.
- Published
- 2016
22. Structure-Based Prediction of Potential Binding and Nonbinding Peptides to HIV-1 Protease
- Author
-
Celia A. Schiffer, Turkan Haliloglu, and Nese Kurt
- Subjects
Models, Molecular ,Polyproteins ,Macromolecular Substances ,medicine.medical_treatment ,Biophysics ,Gene Products, gag ,Gene Products, pol ,Peptide ,Plasma protein binding ,Biophysical Theory and Modeling ,Substrate Specificity ,Motion ,Structure-Activity Relationship ,HIV-1 protease ,HIV Protease ,Enzyme Stability ,medicine ,Electrochemistry ,Computer Simulation ,Binding site ,chemistry.chemical_classification ,Protease ,Binding Sites ,biology ,Active site ,Enzyme Activation ,Biochemistry ,chemistry ,Energy Transfer ,Models, Chemical ,biology.protein ,Threading (protein sequence) ,Protein Binding - Abstract
HIV-1 protease is a major drug target against AIDS as it permits viral maturation by processing the gag and pol polyproteins of the virus. The cleavage sites in these polyproteins do not have obvious sequence homology or a binding motif and the specificity of the protease is not easily determined. We used various threading approaches, together with the crystal structures of substrate complexes which served as template structures, to study the substrate specificity of HIV-1 protease with the aim of obtaining a better differentiation between binding and nonbinding sequences. The predictions from threading improved when distance-dependent interaction energy functions were used instead of contact matrices. To rank the peptides and properly account for the peptide's conformation in the total energy, the results from using short-range potentials on multiple template structures were averaged. Finally, a dynamic threading approach is introduced which is potentially useful for cases when there is only one template structure available. The conformational energy of the peptide—especially the term accounting for the side chains—was found to be important in differentiating between binding and nonbinding sequences. Hence, the substrate specificity, and thus the ability of the virus to mature, is affected by the compatibility of the substrate peptide to fit within the limited conformational space of the active site groove.
- Published
- 2003
- Full Text
- View/download PDF
23. Substrate Shape Determines Specificity of Recognition for HIV-1 Protease
- Author
-
Ellen A. Nalivaika, Celia A. Schiffer, and Moses Prabu-Jeyabalan
- Subjects
Protease ,biology ,Stereochemistry ,Hydrogen bond ,medicine.medical_treatment ,Cleavage (embryo) ,Protein structure ,HIV-1 protease ,Biochemistry ,Structural Biology ,Hydrolase ,biology.protein ,medicine ,Binding site ,Molecular Biology ,Peptide sequence - Abstract
The homodimeric HIV-1 protease is the target of some of the most effective antiviral AIDS therapy, as it facilitates viral maturation by cleaving ten asymmetric and nonhomologous sequences in the Gag and Pol polyproteins. Since the specificity of this enzyme is not easily determined from the sequences of these cleavage sites alone, we solved the crystal structures of complexes of an inactive variant (D25N) of HIV-1 protease with six peptides that correspond to the natural substrate cleavage sites. When the protease binds to its substrate and buries nearly 1000 A2 of surface area, the symmetry of the protease is broken, yet most internal hydrogen bonds and waters are conserved. However, no substrate side chain hydrogen bond is conserved. Specificity of HIV-1 protease appears to be determined by an asymmetric shape rather than a particular amino acid sequence.
- Published
- 2002
24. Curling of Flap Tips in HIV-1 Protease as a Mechanism for Substrate Entry and Tolerance of Drug Resistance
- Author
-
Walter R. P. Scott and Celia A. Schiffer
- Subjects
Models, Molecular ,Conformational change ,Protein Conformation ,Viral protein ,Phenylalanine ,medicine.medical_treatment ,Static Electricity ,Glycine ,Ligands ,medicine.disease_cause ,Substrate Specificity ,Conserved sequence ,Methionine ,Protein structure ,HIV Protease ,HIV-1 protease ,Structural Biology ,medicine ,Humans ,Computer Simulation ,Isoleucine ,Binding site ,Nuclear Magnetic Resonance, Biomolecular ,Molecular Biology ,Conserved Sequence ,Binding Sites ,Protease ,biology ,Active site ,Drug Resistance, Microbial ,Peptide Fragments ,Biochemistry ,HIV-1 ,Mutagenesis, Site-Directed ,Solvents ,biology.protein ,Biophysics ,Thermodynamics - Abstract
Background: The human immunodeficiency virus type 1 (HIV-1) protease is an essential viral protein that is a major drug target in the fight against Acquired Immune Deficiency Syndrome (AIDS). Access to the active site of this homodimeric enzyme is gained when two large flaps, one from each monomer, open. The flap movements are therefore central to the function of the enzyme, yet determining how these flaps move at an atomic level has not been experimentally possible. Results: In the present study, we observe the flaps of HIV-1 protease completely opening during a 10 ns solvated molecular dynamics simulation starting from the unliganded crystal structure. This movement is on the time scale observed by Nuclear Magnetic Resonance (NMR) relaxation data. The highly flexible tips of the flaps, with the sequence Gly-Gly-Ile-Gly-Gly, are seen curling back into the protein and thereby burying many hydrophobic residues. Conclusions: This curled-in conformational change has never been previously described. Previous models of this movement, with the flaps as rigid levers, are not consistent with the experimental data. The residues that participate in this hydrophobic cluster as a result of the conformational change are highly sensitive to mutation and often contribute to drug resistance when they do change. However, several of these residues are not part of the active site cavity, and their essential role in causing drug resistance could possibly be rationalized if this conformational change actually occurs. Trapping HIV-1 protease in this inactive conformation would provide a unique opportunity for future drug design.
- Published
- 2000
25. Comparison of psychosocial adaptation of advanced stage Hodgkin’s disease and acute leukemia survivors
- Author
-
E. Henderson, A. Schilling, George P. Canellos, Raymond B. Weiss, Alice B. Kornblith, Celia A. Schiffer, Richard T. Silver, Jimmie C. Holland, Louis F. Diehl, Robert J. Mayer, Enid Zuckerman, James E. Herndon, S. Wolchok, Donna B. Greenberg, Bruce A. Peterson, M R Cooper, David Cella, and E. Cherin
- Subjects
Pediatrics ,medicine.medical_specialty ,Acute leukemia ,business.industry ,Nausea ,Hematology ,Odds ratio ,Profile of mood states ,Family life ,Oncology ,Quality of life ,medicine ,medicine.symptom ,Psychiatry ,business ,Sexual function ,Psychosocial - Abstract
Background: The purpose of this study was to compare the long-term psychosocial adaptation of Hodgkin's disease and adult acute leukemia survivors. Patients and methods: Two hundred seventy-three Hodgkin's disease (HD) and 206 adult acute leukemia (AL) survivors were interviewed by telephone concerning their psychosocial adjustment and problems they attributed to having been treated for cancer, using identical research procedures and a common set of instruments. The following measures were used: Psychosocial Adjustment to Illness Scale (PAIS); Brief Symptom Inventory (BSI); current Conditioned Nausea and Vomiting triggered by treatment-related stimuli (CNVI); Indices of Employment, Insurance and Sexual Problems Attributed to Cancer; Negative Socioeconomic Impact of Cancer Index (NSI). All participants had been treated on one of nine Hodgkin's disease or 13 acute leukemia Cancer and Leukemia Group B (CALGB) clinical trials from 1966-1988, and had been off treatment for one year or more (mean years: HD = 5.9; AL = 5.6). Results: HD survivors' risk of having a high distress score on the BSI was almost twice that found for AL survivors (odds ratio = 1.90), with 21% of HD vs. 14% of AL survivors (P < 0.05) having scores that were 1.5 standard deviations above the norm, suggestive of a possible psychiatric disorder. HD survivors reported greater fatigue (POMS Fatigue, P = 0.01; Vigor Subscales, P = 0.001). greater conditioned nausea (CNVI, P < 0.05), greater impact of cancer on their family life (PAIS Domestic Environment, P = 0.004) and poorer sexual functioning (PAIS Sexual Relationships. P = 0.0001), than AL survivors. Conclusions: Treatment-related issues may have placed HD survivors at a greater risk for problems in long-term adaptation than AL survivors.
- Published
- 1998
26. Using Molecular Dynamics to Investigate Substrate Recognition and Co-evolution in HIV-1 Protease
- Author
-
Ayşegül Özen, Celia A. Schiffer, and Turkan Haliloglu
- Subjects
chemistry.chemical_classification ,Protease ,Stereochemistry ,medicine.medical_treatment ,Biophysics ,Peptide ,Crystal structure ,Biology ,Cleavage (embryo) ,Molecular dynamics ,symbols.namesake ,HIV-1 protease ,chemistry ,medicine ,biology.protein ,symbols ,Binding site ,van der Waals force - Abstract
Human Immunodeficiency Virus Type-1 (HIV-1) protease recognizes at least ten cleavage sites as its natural substrates. There is little sequence homology between these substrates and they are asymmetric around the cleavage site in both charge and size distribution. Thus, understanding of the molecular determinants of substrate recognition is challenging as well as of great importance in design of effective drugs. The protease-substrate complex crystal structures indicate that substrates occupy a remarkable uniform region within the binding site, which has been termed as the substrate envelope. Nevertheless, protein activity is intimately related to the dynamics, from local to global motion of the structure. To this end, an elaborated analysis on both structural and dynamic features of seven HIV-1 protease-substrate complexes have been carried out by molecular dynamics (MD) simulations. Conformations of the complex structures in time were analyzed with respect to the interaction of substrate with protease in terms of the substrate volume, changes in van der Waals contacts between the two, and dynamics of both substrate and protease. Co-evolution of substrate peptides with the drug-resistant protease variants was also analyzed. Similar analysis to those in wild-type complex structures were done for MD simulations for p1-p6 substrates (wild-type and LP1'F) in complex with protease variants (D30N, N88D, and D30N/N88D). The substrate recognition was observed to be an interdependent event and the recognition mechanism may not be the same for all natural substrates. The dynamic substrate envelope was found to be smaller than the crystal structures suggest. The substrate recognition is altered when there is drug resistance and this alteration is compensated by co-evolution. The results reveal that conservation of the peptide conformational preferences and dynamic behavior of the complex structure appears to be important for substrate recognition.
- Published
- 2009
- Full Text
- View/download PDF
27. Structural Insights into Calmodulation of Neuronal KCNQ Channels
- Author
-
William R. Kobertz, Robert O. Blaustein, Shivender M.D. Shandilya, Karen Mruk, and Celia A. Schiffer
- Subjects
chemistry.chemical_classification ,Physics::Biological Physics ,animal structures ,Calmodulin ,biology ,technology, industry, and agriculture ,Biophysics ,Peptide ,Gating ,Potassium channel ,Calcium in biology ,Quantitative Biology::Cell Behavior ,Quantitative Biology::Subcellular Processes ,Molecular dynamics ,Biochemistry ,chemistry ,biology.protein ,Ion channel ,Intracellular - Abstract
Calmodulin (CaM) is a ubiquitous intracellular calcium sensor for many potassium channels. For the voltage-gated KCNQ family of potassium channels, CaM binds to the intracellular C-terminus to mediate channel assembly, trafficking, and gating. Resolution of the isolated crystal structures of CaM associated with peptide fragments from various ion channels has provided some insight into “calmodulation.” Despite the large repository of these structures, structural information about CaM bound to a fully folded ion channel in the membrane is unknown. We determined the location and orientation of CaM binding with respect to the KCNQ2/KCNQ3 (Q2/Q3) ion-conducting pathway using distance restraints from a panel of intracellular tethered blockers. Molecular dynamics simulations of the Q2/Q3-CaM complex position CaM strikingly close to the gate of Q2/Q3. The structure of the Q2/Q3-CaM complex and its implications for calcium-induced calmodulation will be discussed.
- Published
- 2012
28. Erratum to 'Covariation of amino acid positions in HIV-1 protease' [Virology 314 (2003) 536–548]
- Author
-
Ronald Swanstrom, Celia A. Schiffer, and Noah G. Hoffman
- Subjects
chemistry.chemical_classification ,Biochemistry ,HIV-1 protease ,biology ,chemistry ,Virology ,biology.protein ,Amino acid - Published
- 2005
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.