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Molecular basis for the subunit assembly of the primase from an archaeon Pyrococcus horikoshii
Author(s) -
Ito Nobutoshi,
Matsui Ikuo,
Matsui Eriko
Publication year - 2007
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2007.05690.x
Subject(s) - pyrococcus horikoshii , protein subunit , alanine scanning , crystallography , alanine , pyrococcus furiosus , biology , chemistry , biochemistry , stereochemistry , mutant , archaea , gene , mutagenesis , amino acid , enzyme
Archaeal/eukaryotic primases form a heterodimer consisting of a small catalytic subunit (PriS) and a large subunit (PriL). The heterodimer complex synthesizes primer oligoribonucleotides that are required for chromosomal replication. Here, we describe crystallographic and biochemical studies of the N‐terminal domain (NTD) of PriL (PriL NTD ; residues 1–222) that bind to PriS from a hyperthermophilic archaeon, Pyrococcus horikoshii , at 2.9 Å resolution. The PriL NTD structure consists of two subdomains, the helix‐bundle and twisted‐strand domains. The latter is structurally flexible, and is expected to contain a PriS interaction site. Pull‐down and surface plasmon resonance analyses of structure‐based deletion and alanine scanning mutants showed that the conserved hydrophobic Tyr155‐Tyr156‐Ile157 region near the flexible region is the PriS‐binding site, as the Y155A/Y156A/I157A mutation markedly reduces PriS binding, by 1000‐fold. These findings and a structural comparison with a previously reported PriL NTD –PriS complex suggest that the presented alternative conformations of the twisted‐strand domain facilitate the heterodimer assembly.