Extensive deamidation of RNase A inhibits its oligomerization through 3D domain swapping

• Published in: Biochimica et biophysica acta. Proteins and proteomics 2017-01, Vol.1865 (1), p.76-87
• Main Authors: Fagagnini, Andrea, Montioli, Riccardo, Caloiu, Andra, Ribó, Marc, Laurents, Douglas V., Gotte, Giovanni
• Format: Article
• Language: English
• Subjects: 3D domain swapping; Amides - chemistry; Amyloid - chemistry; Animals; Asparagine - chemistry; Asparagine - genetics; Aspartic Acid - chemistry; Aspartic Acid - genetics; Cattle; Enzyme Stability; Glutamine - chemistry; Mutation; Nuclear Magnetic Resonance, Biomolecular; Protein aggregation; Protein deamidation; Protein Domains; Protein Multimerization; Protein oligomers; Ribonuclease A; Ribonuclease, Pancreatic - chemistry; Ribonuclease, Pancreatic - genetics; RNase A oligomers
• ISSN: 1570-9639; 1878-1454
• DOI: 10.1016/j.bbapap.2016.10.008

Bovine pancreatic ribonuclease A (RNase A) is the monomeric prototype of the so-called secretory ‘pancreatic-type’ RNase super-family. Like the naturally domain-swapped dimeric bovine seminal variant, BS-RNase, and its glycosylated RNase B isoform, RNase A forms N- and C-terminal 3D domain-swapped oligomers after lyophilization from acid solutions, or if subjected to thermal denaturation at high protein concentration. All mentioned RNases can undergo deamidation at Asn67, forming Asp or isoAsp derivatives that modify the protein net charge and consequently its enzymatic activity. In addition, deamidation slightly affects RNase B self-association through the 3D domain swapping (3D-DS) mechanism. We report here the influence of extensive deamidation on RNase A tendency to oligomerize through 3D-DS. In particular, deamidation of Asn67 alone slightly decreases the propensity of the protein to oligomerize, with the Asp derivative being less affected than the isoAsp one. Contrarily, the additional Asp and/or isoAsp conversion of residues other than N67 almost nullifies RNase A oligomerization capability. In addition, Gln deamidation, although less kinetically favorable, may affect RNase A self-association. Using 2D and 3D NMR we identified the Asn/Gln residues most prone to undergo deamidation. Together with CD spectroscopy, NMR also indicates that poly-deamidated RNase A generally maintains its native tertiary structure. Again, we investigated in silico the effect of the residues undergoing deamidation on RNase A dimers structures. Finally, the effect of deamidation on RNase A oligomerization is discussed in comparison with studies on deamidation-prone proteins involved in amyloid formation. [Display omitted] •RNase A deamidation affects its oligomerization occurring via 3D domain swapping.•IsoAsp derivatives lower oligomerization more than the corresponding Asp products.•Extensive deamidation almost nullifies RNase A self-association tendency.•It's the first report associating protein deamidation to zero aggregation tendency.