Molecular basis for the subunit assembly of the primase from an archaeon Pyrococcus horikoshii.
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 A 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.
Ito
Nobutoshi
N
Cellular Physiology Laboratory, Discovery Research Institute, RIKEN, Wako, Saitama, Japan.
Matsui
Ikuo
I
Matsui
Eriko
E
D019943:Amino Acid Substitution; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D019915:DNA Primase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D005978:Glutathione / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D043902:Pyrococcus horikoshii / Q000201:enzymology* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013057:Spectrum Analysis; D013329:Structure-Activity Relationship; D020349:Surface Plasmon Resonance
D019843:Archaeal Proteins; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D019915:DNA Primase; D005978:Glutathione