Origin of Dimeric Structure in the Ribonuclease Superfamily

Abstract

To enable application of postgenomic evolutionary approaches to understand the divergence of behavior and function in ribonucleases (RNases), the impact of divergent sequence on the divergence of tertiary and quaternary structure is analyzed in bovine pancreatic and seminal ribonucleases, which differ by 23 amino acids. In a crystal, seminal RNase is a homodimer joined by two "antiparallel" intersubunit disulfide bonds between Cys-31 from one subunit and Cys-32' from the other and having composite active sites arising from the "swap" of residues 1-20 from each subunit. Specialized Edman degradation techniques have completed the structural characterization of the dimer in solution, new cross-linking methods have been developed to assess the swap, and sequence determinants of quaternary structure have been explored by protein engineering using the reconstructed evolutionary history of the protein family as a guide. A single Cys at either position 32 (the first to be introduced during the divergent evolution of the family) or 31 converts monomeric RNase A into a dimer. Even with an additional Phe at position 31, another residue introduced early in the seminal lineage, swap is minimal. A hydrophobic contact formed by Leu-28, however, also introduced early in the seminal lineage, increases the amount of "antiparallel" connectivity of the two subunits and facilitates swapping of residues 1-20. Efficient swapping requires addition of a Pro at position 19, a residue also introduced early in the divergent evolution of the seminal RNase gene. Additional cysteines required for dimer formation are found to slow refolding of the protein through formation of incorrect disulfide bonds, suggesting a paradox in the biosynthesis of the protein. Further studies showed that the dimeric form of seminal RNase known in the crystal is not the only form in vivo, where a substantial amount of heterodimer is known. These data complete the acquisition of the background needed to understand the evolution of new structure, behavior, and function in the seminal RNase family of proteins.

The Role of the Hinge Loop in Domain Swapping

“…Fig. 1 19 -mBS (55°C) is 6°C lower than that of RNase A; the difference is even higher in the mutant containing four substitutions in the hinge region (T m ϭ 53.5°C), despite the greater similarity of its sequence to that of RNase A. In both cases the effect is significantly greater than that produced by the sole substitution of Asn 67 (27).…”

“…The dimers (20 g) in sodium acetate buffer (100 mM, pH 5, 100 l) and DVS (1 l of the 10% solution) were incubated at 30°C (19). This is approximately a 1,000-fold excess of sulfone to each subunit of the protein.…”

“…Ala 19 -BS-RNase data collection was carried out at room temperature up to 2.20-Å resolution. Because of the lower resolution of the diffraction data, a Ser 16 -Thr 17 -Ala 19 -Ala 20 -BS-RNase crystal was cryo-cooled at 100 K by using 18% (v/v) glycerol as cryo-protector, and the diffraction data were collected up to the same resolution (2.20 Å). The two data sets were processed and scaled with the HKL package (21).…”

“…The characterization of the swapping process for Ala 19 -BS-RNase has been already reported (12). Two independent methods have been used to evaluate the swapping tendency of the other mutant.…”

“…In order to clarify the actual role of the hinge sequence in the swapping process of BS-RNase, a homologue-scanning mutagenesis approach has been followed, using as reference the sequence of RNase A. We have prepared two mutants, Ala 19 …”