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Cited by 99Enzyme Structure and FunctionProtein Structure and DynamicsDNA and Nucleic Acid Chemistry

Crystal Structures of an NH2-Terminal Fragment of T4 DNA Polymerase and Its Complexes with Single-Stranded DNA and with Divalent Metal Ions

Wang, J., Yu, P., Lin, T. C., Konigsberg, W. H., Steitz, T. A.·1996·Biochemistry
DOI 10.1021/bi960178r
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Abstract

We report the crystal structure of an NH2-terminal 388-residue fragment of T4 DNA polymerase (protein N388) refined at 2.2 A resolution. This fragment contains both the 3'-5' exonuclease active site and part of the autologous mRNA binding site (J. D. Karam, personal communication). The structure of a complex between the apoprotein N388 and a substrate, p(dT)3, has been refined at 2.5 A resolution to a crystallographic R-factor of 18.7%. Two divalent metal ion cofactors, Zn(II) and Mn(II), have been located in crystals of protein N388 which had been soaked in solutions containing Zn(II), Mn(II), or both. The structure of the 3'-5' exonuclease domain of protein N388 closely resembles the corresponding region in the Klenow fragment despite minimal sequence identity. The side chains of four carboxylate residues that serve as ligands for the two metal ions required for catalysis are located in geometrically equivalent positions in both proteins with a rms deviation of 0.87 A. There are two main differences between the 3'-5' exonuclease active site regions of the two proteins: (I) the OH of Tyr-497 in the Klenow fragment interacts with the scissile phosphate in the active site whereas the OH of the equivalent tyrosine (Tyr-320) in protein N388 points away from the active center; (II) different residues form of the binding pocket for the 3'-terminal bases of the substrate. In the protein N388 complex the 3'-terminal base of p(dT)3 is rotated approximately 60 degrees relative to the position that the corresponding base occupies in the p(dT)3 complex with the Klenow fragment. Finally, a separate domain (residues 1-96) of protein N388 may be involved in mRNA binding that results in translational regulation of T4 DNA polymerase (Pavlov & Karam, 1994).

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  • Zn2+ is an interesting alternative to Mg2+ as a catalytic metal ion for RNA polymerization because many protein enzymes use a bound Zn2+ ion in their active sites.

    “Zn 2+ is an interesting alternative to Mg 2+ as a catalytic metal ion for RNA polymerization, in large part because many protein enzymes, such as the DNA polymerase 3 ' -5 ' exonuclease domain, use a bound Zn 2+ ion in their active sites [64].”

    From The eightfold path to non-enzymatic RNA replication (2012) · cites this paper as [64]

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Authors
Wang, J., Yu, P., Lin, T. C., Konigsberg, W. H., & Steitz, T. A.
Affiliations
  • Howard Hughes Medical Institute (US)
  • Department of Molecular Biophysics and Biochemistry, Department of Chemistry, and Howard Hughes Medical Institute, Yale University, New Haven, Connecticut 06520-8114
ORCID
Venue
Biochemistry
Publisher
American Chemical Society (ACS)
Published
1996-01-01
Volume
35
Issue
25
Pages
8110-8119
ISSN
  • 0006-2960 (ISSN-L)
  • 1520-4995
DOI
10.1021/bi960178r
OpenAlex
W2035834598
Cited by
99
References
12
Citations by year
2026: 1 · 2025: 2 · 2024: 1 · 2023: 1 · 2022: 2 · 2021: 2 · 2020: 2 · 2019: 5 · 2018: 3 · 2017: 2 · 2016: 5 · 2015: 2 · 2014: 1 · 2013: 2 · 2012: 3
FWCI
8.77
Type
journal article
Language
en
Open access
closed
Locations
  • Biochemistry (publishedVersion)
  • PubMed (publishedVersion)
Funders
  • National Institutes of Health (5r01gm012607-30)
  • National Institute of General Medical Sciences (GM12607-31)
Topics
  • Physical Sciences › Materials Science › Materials Chemistry › Enzyme Structure and Function
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA and Nucleic Acid Chemistry
Keywords
T4 DNA polymerase, Klenow fragment, divalent metal ions, zinc, manganese, single-stranded DNA, mRNA binding, translational regulation, protein structure, carboxylate ligands
MeSH terms
Amino Acid Sequence, Binding Sites, Cations, Divalent, DNA-Directed DNA Polymerase, DNA, Single-Stranded, Exodeoxyribonucleases, Manganese, Models, Molecular, Molecular Sequence Data, Peptide Fragments, Protein Binding, Viral Proteins, Zinc, Crystallography, X-Ray

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