Cataract-prone variants of γD-crystallin populate a conformation with a partially unfolded N-terminal domain under native conditions

Abstract

Human {gamma}D-crystallin, a monomeric protein abundant in the eye lens nucleus, must remain stably folded for an individuals entire lifetime to avoid aggregation and protein deposition-associated cataract formation. {gamma}D-crystallin contains two homologous domains, an N-terminal domain (NTD) and a C-terminal domain (CTD), which interact via a hydrophobic interface. A number of familial mutations in the gamma crystallin gene are linked to congenital early-onset cataract, most of which result in amino acid changes in the NTD. Several of these, such as V75D and W42R, are known to populate intermediates that, under partially denaturing conditions, possess a natively folded CTD and a completely unfolded NTD, with studies on W42R showing further evidence for a minor population of an intermediate under native conditions. We employed hydrogen-deuterium exchange mass spectrometry (HDX-MS) to probe the structural and energetic features of variants of {gamma}D-crystallin under both native and partially denaturing conditions. For V75D and W42R, we identify a species under native conditions that retains partial structure in the NTD and is structurally and energetically distinct from the intermediate populated under partially denaturing conditions. Residues at the NTD-CTD interface play crucial roles in stabilizing this intermediate, and disruption of interface contacts either by amino acid substitution or partial denaturation permits direct observation of two intermediates at the same time. The newly identified intermediate exposes hydrophobic amino acids that are buried in both the folded full-length protein and in the proteins stable isolated domains. Such non-native exposure of a hydrophobic patch may play an important role in cataract formation. Significance StatementHuman {gamma}D-crystallin, which plays a structural role in the eye lens, is a long-lived protein that must remain folded for an individuals entire lifetime to avoid aggregation and protein deposition - associated cataract formation. By using hydrogen-deuterium exchange mass spectrometry, we demonstrate that two cataract-associated variants of {gamma}D-crystallin populate an intermediate with partial structure along the interface between its two domains under native conditions. In these intermediates, hydrophobic amino acids that are normally buried in the N-terminal domains native folded structure become exposed, possibly leading to aggregation and cataract formation. Our findings illustrate the importance of studying a proteins energy landscapes under conditions that are close to physiological.