Identification of crucial hydrogen-bonding residues for the interaction of herpes simplex virus DNA polymerase subunits via peptide display, mutational, and calorimetric approaches

被引:30
作者
Bridges, KG
Chow, CS
Coen, DM
机构
[1] Harvard Univ, Sch Med, Dept Biol Chem & Mol Pharmacol, Boston, MA 02115 USA
[2] Harvard Univ, Sch Med, Comm Virol, Boston, MA 02115 USA
关键词
D O I
10.1128/JVI.75.11.4990-4998.2001
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
The catalytic subunit, Pol, of herpes simplex virus DNA polymerase interacts via its extreme C terminus with the processivity subunit, UL42. This interaction is critical for viral replication and thus a potential target for antiviral drug action. To investigate the Pol-binding region on UL42, we engineered UL42 mutations but also used random peptide display to identify artificial ligands of the Pol C terminus. The latter approach selected ligands with homology to residues 171 to 176 of UL42, Substitution of glutamine 171 with alanine greatly impaired binding to Pol and stimulation of long-chain DNA synthesis by Pol, identifying this residue as crucial for subunit interactions. To study these interactions quantitatively, we used isothermal titration calorimetry and wild-type and mutant forms of Pol-derived peptides and UL42. Each of three peptides corresponding to either the Last 36, 27, or 18 residues of Pol bound specifically to UL42 in a 1:1 complex with a dissociation constant of 1 to 2 muM. Thus, the last 18 residues suffice for most of the binding energy, which was due mainly to a change in enthalpy. Substitutions at positions corresponding to Pol residue 1228 or 1229 or at UL42 residue 171 abolished or greatly reduced binding. These residues participate in hydrogen bonds observed in the crystal structure of the C terminus of Pol bound to UL42. Thus, interruption of these few bonds is sufficient to disrupt the interaction, suggesting that small molecules targeting the relevant side chains could interfere with Pol-UL42 binding.
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页码:4990 / 4998
页数:9
相关论文
共 22 条
  • [1] Ausubel FM, 1995, SHORT PROTOCOLS MOL
  • [2] CLONING, SEQUENCING, AND FUNCTIONAL-CHARACTERIZATION OF THE 2 SUBUNITS OF THE PSEUDORABIES VIRUS DNA POLYMERASE HOLOENZYME - EVIDENCE FOR SPECIFICITY OF INTERACTION
    BERTHOMME, H
    MONAHAN, SJ
    PARRIS, DS
    JACQUEMONT, B
    EPSTEIN, AL
    [J]. JOURNAL OF VIROLOGY, 1995, 69 (05) : 2811 - 2818
  • [3] Secondary structure and structure-activity relationships of peptides corresponding to the subunit interface of herpes simplex virus DNA polymerase
    Bridges, KG
    Hua, QX
    Brigham-Burke, MR
    Martin, JD
    Hensley, P
    Dahl, CE
    Digard, P
    Weiss, MA
    Coen, DM
    [J]. JOURNAL OF BIOLOGICAL CHEMISTRY, 2000, 275 (01) : 472 - 478
  • [4] MUTATIONS THAT SPECIFICALLY IMPAIR THE DNA-BINDING ACTIVITY OF THE HERPES-SIMPLEX VIRUS PROTEIN UL42
    CHOW, CS
    COEN, DM
    [J]. JOURNAL OF VIROLOGY, 1995, 69 (11) : 6965 - 6971
  • [5] DIGARD P, 1990, J BIOL CHEM, V265, P17393
  • [6] FUNCTIONAL-ANALYSIS OF THE HERPES-SIMPLEX VIRUS UL42 PROTEIN
    DIGARD, P
    CHOW, CS
    PIRRIT, L
    COEN, DM
    [J]. JOURNAL OF VIROLOGY, 1993, 67 (03) : 1159 - 1168
  • [7] SPECIFIC-INHIBITION OF HERPES-SIMPLEX VIRUS-DNA POLYMERASE BY HELICAL PEPTIDES CORRESPONDING TO THE SUBUNIT INTERFACE
    DIGARD, P
    WILLIAMS, KP
    HENSLEY, P
    BROOKS, IS
    DAHL, CE
    COEN, DM
    [J]. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1995, 92 (05) : 1456 - 1460
  • [8] THE EXTREME-C TERMINUS OF HERPES-SIMPLEX VIRUS-DNA POLYMERASE IS CRUCIAL FOR FUNCTIONAL INTERACTION WITH PROCESSIVITY FACTOR-UL42 AND FOR VIRAL REPLICATION
    DIGARD, P
    BEBRIN, WR
    WEISSHART, K
    COEN, DM
    [J]. JOURNAL OF VIROLOGY, 1993, 67 (01) : 398 - 406
  • [9] THE HERPES-SIMPLEX VIRUS TYPE-1 DNA-POLYMERASE ACCESSORY PROTEIN, UL42, CONTAINS A FUNCTIONAL PROTEASE-RESISTANT DOMAIN
    HAMATAKE, RK
    BIFANO, M
    TENNEY, DJ
    HURLBURT, WW
    CORDINGLEY, MG
    [J]. JOURNAL OF GENERAL VIROLOGY, 1993, 74 : 2181 - 2189
  • [10] Sensing the heat: The application of isothermal titration calorimetry to thermodynamic studies of biomolecular interactions
    Ladbury, JE
    Chowdhry, BZ
    [J]. CHEMISTRY & BIOLOGY, 1996, 3 (10): : 791 - 801