CRYSTAL STRUCTURE OF RECOMBINANT PORCINE S100A12 AT 1.38Å RESOLUTION

FQ5

Pinheiro, MP(1); Garcia, AF(2); Bordon, KCF(1); Araújo, APU(2); Nonato, MC(1)

(1) Laboratório de Cristalografia de Proteínas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, FCFRP-USP, Ribeirão Preto, SP, Brazil;

(2) Instituto de Física de São Carlos, Universidade de São Paulo, IFSC-USP, São Carlos, SP, Brazil.

Porcine S100A12 (also known as calgranulin C) is a member of the S100 proteins, family of small acidic calcium-binding proteins characterized by the presence of two EF-hand motifs. These proteins are involved in many cellular events such as the regulation of protein phosphorylation, enzymatic activity, protein–protein interaction, Ca²⁺ homeostasis, inflammatory processes and intermediate filament polymerization. Here, we report the crystal structure of porcine S100A12 solved at 1.38Å resolution. The structure has been solved by single anomalous scattering (SAD) techniques using the iodine anomalous signal. For our crystallographic studies, S100A12 has been crystallized using vapour-diffusion techniques in sitting drops at 295 K using ammonium sulfate as precipitant. The growth of prismatic crystals was visible after one day with crystal size reaching up to 0.3 x 0.5 x 0.5 mm. Iodide derivative crystals were obtained by using rapid cryo-soaking techniques. Diffraction data were collected from frozen crystals (at 100 K), using a wavelength of 1.30Å for native crystals and 1.54Å for iodide derivatives crystals. X-ray diffraction data were collected at the protein crystallography beam line W01B-MX2 of the Brazilian Synchrotron Light Laboratory, LNLS, Campinas, Brazil, using a MarMosaic 225 detector. Data processing and scaling were performed using the programs MOSFLM and SCALA from the CCP4 package. CAGC native crystals diffract to 1.38Å resolution and belong to the triclinic space group P3₁21, with unit-cell parameters a=b=51.70 and c=65.93Å. Data from iodide derivative crystals were collected up to 1.7Å resolution. Derivative crystals belong to the same space group as native crystals with similar unit cell parameters. We used the AutoSol tool from the PHENIX package to setting up inputs, analyzing and scaling the data, finding heavy-atom (anomalously-scattering atom) sites, scoring of heavy-atom solutions, phasing, density modification, and preliminary model-building and refinement. The final structure refinement was performed using the program REFMAC (CCP4), with the native diffraction data set at 1.38Å resolution. The final model presented an Rfactor and Rfree of 17.5% and 19.7%, respectively, with good overall stereochemistry. The structure reveals important differences in conformation and secondary structure compared to the human protein which will be used to correlate the difference in function and specificity observed for this class of proteins in different species (FAPESP).