Influence of intermolecular contacts on the structure of recombinant prolidase fromThermococcus sibiricus

Abstract

Prolidases are peptidases that are specific for dipeptides with proline as the second residue. The structure of recombinant prolidase from the hyperthermophilic archaeonThermococcus sibiricus(Tsprol) was determined at 2.6 Å resolution. The homodimer ofTsprol is characterized by a complete lack of interactions between the N- and C-terminal domains of the two subunits and hence can be considered to be the most open structure when compared with previously structurally studied prolidases. This structure exists owing to intermolecular coordination bonds between cadmium ions derived from the crystallization solution and histidine residues of a His tag and aspartate and glutamate residues, which link the dimers to each other. This linking leads to the formation of a crystal with a loose packing of protein molecules and low resistance to mechanical influence and temperature increase.

Structures and activities of widely conserved small prokaryotic aminopeptidases‐P clarify classification of M24B peptidases

“…Structures were solved by molecular replacement (MR) using PHASER suite on the CCP4 platform. The MR model was prepared by Chainsaw using the putative prolidase from T. sibiricus (PDB ID: 4FKC) . Automated model building was performed using phenix.autobuild .…”

“…The MR model was prepared by Chainsaw 32 using the putative prolidase from T. sibiricus (PDB ID: 4FKC). 33 Automated model building was performed using phenix.autobuild. 34 Subsequently, the model was completed by iterative rounds of manual model building using COOT 35 and refinements using REFMAC5 36 and phenix.refine.…”

Structure of recombinant prolidase fromThermococcus sibiricusin space groupP21221

“…The Tsib_0821 gene was produced via PCR amplification of the recombinant plasmid pQE80L_TSIB0821 (Trofimov et al, 2012) with the primers Tsprol-1_For (5 0 -GCCCATGGAT-TACAAGAGAAGGATTCATAAG-3 0 ) and Tsprol-1_Rev (5 0 -TTTGTCGACCTATAGCGTGATCAATTCCCTATC-3 0 ) containing NcoI and SalI restriction sites, respectively. The resulting PCR product encoding full-length Tsprol-1 was digested by these restriction enzymes and cloned into a polylinker of a modified pET-22b vector (Novagen, Darmstadt, Germany).…”

“…Structural and functional properties of two thermostable homologues, PH1149 and PH0974, from P. horikoshii were subsequently determined (Jeyakanthan et al, 2009;Theriot et al, 2010). Recently, we reported the crystal structure of the prolidase Tsprol from the thermophilic organotrophic archaeon Thermococcus sibiricus (Trofimov et al, 2012). The crystallized protein contained a 6ÂHis-tag sequence at the N-terminus, and analysis of the solved structure demonstrated the contribution of amino-acid residues from the 6ÂHis tag and cadmium ions from the crystallization solution to the folding of the Tsprol dimers.…”

Crystallization and preliminary X-ray diffraction analysis of Xaa-Pro dipeptidase fromXanthomonas campestris

“…The closest sequence homologues of the XPD48 and XPD43 proteins of X. campestris in the PDB are the XPDs from Alteromonas sp. strain JD6.5 (PDB entry 3l24; 47% identity) and Thermococcus sibiricus (PDB entry 4fkc; 29% identity; Trofimov et al, 2012), respectively.…”