Expression, purification, and crystallography of the conserved methionine-rich domain of human signal recognition particle 54 kDa protein

Protein SRP54 is an essential component of eukaryotic signal recognition pa rticle (SRP). The methionine-rich M-domain (SRP54M or 54M) interacts with t he SRP RNA and is also involved in the binding to signal peptides of secret ory proteins during their targeting to cellular membranes. To gain insight into the molecular details of SRP-mediated protein targeting, we studied th e human 54M polypeptide. The recombinant human protein was expressed succes sfully in Escherichia coli and was purified to homogeneity. Our studies det ermined the sites that were susceptible to limited proteolysis, with the go al to design smaller functional mutant derivatives that lacked nonessential amino acid residues from both termini. Of the four polypeptides produced b y V8 protease or chymotrypsin, 54MM-2 was the shortest (120 residues; M-r = 13,584.8), but still contained the conserved amino acids suggested to asso ciate with the signal peptide or the SRP RNA. 54MM-2 was cloned, expressed, purified to homogeneity, and was shown to bind human SRP RNA in the presen ce of protein SRP19, indicating that it was functional. Highly reproducible conditions for the crystallization of 54MM-2 were established. Examination of the crystals by X-ray diffraction showed an orthorhombic unit cell of d imensions a = 29.127 Angstrom, b = 63.693 Angstrom, and c = 129.601 Angstro m, in space group P2(1)2(1)2(1), with reflections extending to at least 2.0 Angstrom.