The unswapped chain of bovine seminal ribonuclease: Crystal structure of the free and liganded monomeric derivative.
Bovine seminal ribonuclease, a homodimeric enzyme joined covalently by two interchain disulphide bonds, is an equilibrium mixture of two conformational isomers, MxM and M=M. The major form, MxM, whose crystal structure has been previously determined at 1.9 A resolution, presents the swapping of the N-terminal segments (residues 1-15) and composite active sites formed by residues of different chains. The three-dimensional domain swapping does not occur in the M=M form. The different fold of each N-terminal tail is directed by the hinge loop (residue 16-22) connecting the swapping domain to the body of the protein. Reduction and alkylation of interchain disulphide bridges produce a monomeric derivative and a noncovalent swapped dimer, which are both active. The free and nucleotide-bound forms of the monomer have been crystallized at an alkaline pH and refined at 1.45 and 1.65 A resolution, respectively. In both cases, the N-terminal fragment is folded on the main body of the protein to produce an intact active site and a chain architecture very similar to that of bovine pancreatic ribonuclease. In this new fold of the seminal chain, the hinge loop is disordered. Despite the difference between the tertiary structure of the monomer and that of the chains in the MxM form, the active sites of the two enzymes are virtually indistinguishable. Furthermore, the structure of the liganded enzyme represents the first example of a ribonuclease complex studied at an alkaline pH and provides new information on the binding of a nucleotide when the catalytic histidines are deprotonated.
Sica
Filomena
F
Dipartimento di Chimica, Università degli Studi di Napoli Federico II, Napoli, Italy.
Di Fiore
Anna
A
Zagari
Adriana
A
Mazzarella
Lelio
L
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D018360:Crystallography, X-Ray; D004722:Endoribonucleases / Q000737:chemistry* / Q000378:metabolism; D008024:Ligands; D008958:Models, Molecular; D009711:Nucleotides / Q000378:metabolism
D008024:Ligands; D009711:Nucleotides; D004722:Endoribonucleases; C032523:ribonuclease SPL