title: Interactions crucial for three-dimensional domain swapping in the HP-RNase variant PM8.
abstract: The structural determinants that are responsible for the formation of higher order associations of folded proteins remain unknown. We have investigated the role on the dimerization process of different residues of a domain-swapped dimer human pancreatic ribonuclease variant. This variant is a good model to study the dimerization and swapping processes because dimer and monomer forms interconvert, are easily isolated, and only one dimeric species is produced. Thus, simple models for the swapping process can be proposed. The dimerization (dissociation constant) and swapping propensity have been studied using different variants with changes in residues that belong to different putative molecular determinants of dimerization. Using NMR spectroscopy, we show that these mutations do not substantially alter the overall conformation and flexibility, but affect the residue level stability. Overall, the most critical residues for the swapping process are those of one subunit that interact with the hinge loop of another one-subunit residue, stabilizing it in a conformation that favors the interchange. Tyr(25), Gln(101), and Pro(19), with Asn(17), Ser(21), and Ser(23), are found to be the most significant; notably, Glu(103) and Arg(104), which were postulated to form salt bridges that would stabilize the dimer, are not critical for dimerization.
authors.affiliation: Laboratori d'Enginyeria de Proteïnes, Departament de Biologia, Facultat de Ciències, Universitat de Girona, Campus de Montilivi, Maria Aurèlia Campmany, 69 E-17071 Girona, Spain.
authors.forename: Pere
authors.initials: P
authors.lastname: Tubert
authors.forename: Douglas V
authors.initials: DV
authors.lastname: Laurents
authors.forename: Marc
authors.initials: M
authors.lastname: Ribó
authors.forename: Marta
authors.initials: M
authors.lastname: Bruix
authors.forename: Maria
authors.initials: M
authors.lastname: Vilanova
authors.forename: Antoni
authors.initials: A
authors.lastname: Benito
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D004795:Enzyme Stability; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D050505:Mutant Proteins / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000378:metabolism*; D013816:Thermodynamics
chemical_list: D000596:Amino Acids; D050505:Mutant Proteins; D021122:Protein Subunits; D012259:Ribonuclease, Pancreatic