Cryptic genetic variations of alanine:glyoxylate aminotransferase shape its fitness and dynamics

Abstract

Genetic variations expand the conformational landscape of proteins and may underlie cryptic functions able to influence protein adaptability under unfavorable conditions. Cryptic functions usually associate with regions of increased frustration or even intrinsically disordered, whose role as drivers of innovation is increasingly being recognized. Hence, the balance between protein stability and controlled disorder translates in the dichotomy between conservation and innovability, and should be regarded as the more comprehensive measure of protein fitness. In this context, understanding how genetic variations affect protein fitness is not trivial, since cryptic functions behind frustrated regions are not easily detectable. Herein, we used as model the pyridoxal 5-phosphate (PLP)-dependent enzyme alanine:glyoxylate aminotransferase (AGT), which is present as a common major allelic form (AGT-Ma) and a minor polymorphic form (AGT-Mi) expressed in 20% of Caucasian population and considered a lower limit of AGT fitness. By solving the structure of AGT-Mi, we could show that three distinct regions, that are structured in AGT-Ma, are disordered in AGT-Mi. Molecular dynamics shows that AGT-Mi samples more flexible conformations than AGT-Ma, supporting a plasticity effect propagated to all the structure. In-depth biochemical characterisation of mutants from a small library of variants encompassing the three regions reproduces the fitness window between AGT-Ma and AGT-Mi. Cellular studies highlight the consequences of this plasticity at functional level and suggest the existence of cryptic functions likely related to protein-protein interactions. These results establish that naturally-occurring genetic variations tip the balance between protein stability and frustration to encode cryptic functions that expand the potential innovability of the protein.