Crystallization and X-ray diffraction studies of crustacean proliferating cell nuclear antigen

Abstract

Proliferating cell nuclear antigen (PCNA), a member of the sliding clamp family of proteins, interacts specifically with DNA replication and repair proteins through a small peptide motif called the PCNA-interacting protein or PIP box. PCNA is recognized as one of the key proteins involved in DNA metabolism. In the present study, the recombinant PCNA from Litopenaeus vannamei (LvPCNA) was heterologously overexpressed and purified using metal ion-affinity chromatography. Crystals suitable for diffraction grew overnight using the hanging-drop vapour-diffusion method. LvPCNA crystals belong to space group C2 with unit-cell parameters a=144.6, b=83.4, c=74.3 Å, β=117.6°. One data set was processed to 3 Å resolution, with an overall Rmeas of 0.09 and a completeness of 93.3%. Initial phases were obtained by molecular replacement using a homology model of LvPCNA as the search model. Refinement and structural analysis are underway. This report is the first successful crystallographic analysis of a marine crustacean decapod shrimp (L. vannamei) proliferating cell nuclear antigen.

Crystal Structure of the Shrimp Proliferating Cell Nuclear Antigen: Structural Complementarity with WSSV DNA Polymerase PIP-Box

“…LvPCNA eluted in a complex of approximately 90 kDa, indicating that this protein assembles as a trimer in solution [27]. Accordingly to this previous result the molecular replacement found a trimer in the asymmetric unit.…”

“…To determine the quaternary structure of LvPCNA we run this purified protein at 1mg/ml into a Superdex 200 size-exclusion chromatography column and compared its elution profile with known molecular-mass standards. LvPCNA eluted in a complex of approximately 90 kDa, indicating that this protein assembles as a trimer in solution [27] . Accordingly to this previous result the molecular replacement found a trimer in the asymmetric unit.…”

“…Thin hexagonal shaped crystals of approximately 0.1×0.6 mm were suitable for X-ray diffraction. The Lv PCNA crystal belonged to the C2 space group with unit-cell parameters a  = 144.6 Å, b  = 83.4 Å, c  = 74.3 Å, β = 117.6° [27].…”

“…Metal affinity chromatography method was used for purification. Detailed description of overexpression, purification and Lv PCNA crystallization methods were previously reported [27] .…”

“…In vitro , this polymerase had a low processivity, although the presence of a PIP-box in its sequence and the absence of putative processivity factors in the virus genome suggest that it utilizes a host processivity factor [24] , [25] , [26] . We have recently reported the cDNA sequence, recombinant overexpression, purification and crystallization of the shrimp Litopenaeus vannamei PCNA [25] , [27] . Moreover, others and ourselves have reported its gene expression during viral infection [25] , [28] , [29] , [30] .…”