Deletion of internal structured repeats increases the stability of a leucine-rich repeat protein, YopM

Abstract

Mapping the stability distributions of proteins in their native folded states provides a critical link between structure, thermodynamics, and function. Linear repeat proteins have proven more amenable to this kind of mapping than globular proteins. C-terminal deletion studies of YopM, a large, linear leucine-rich repeat (LRR) protein, show that stability is distributed quite heterogeneously, yet a high level of cooperativity is maintained [1]. Key components of this distribution are three interfaces that strongly stabilize adjacent sequences, thereby maintaining structural integrity and promoting cooperativity. To better understand the distribution of interaction energy around these critical interfaces, we studied internal (rather than terminal) deletions of three LRRs in this region, including one of these stabilizing interfaces. Contrary to our expectation that deletion of structured repeats should be destabilizing, we find that internal deletion of folded repeats can actually stabilize the native state, suggesting that these repeats are destabilizing, although paradoxically, they are folded in the native state. We identified two residues within this destabilizing segment that deviate from the consensus sequence at a position that normally forms a stacked leucine ladder in the hydrophobic core. Replacement of these nonconsensus residues with leucine is stabilizing. This stability enhancement can be reproduced in the context of nonnative interfaces, but it requires an extended hydrophobic core. Our results demonstrate that different LRRs vary widely in their contribution to stability, and that this variation is context-dependent. These two factors are likely to determine the types of rearrangements that lead to folded, functional proteins, and in turn, are likely to restrict the pathways available for the evolution of linear repeat proteins.

Highly polarized C-terminal transition state of the leucine-rich repeat domain of PP32 is governed by local stability

“…In contrast, HX studies observe unfolding reactions in the context of the folded state, such that structural modules (foldons) (47) can stabilize one another. Work on consensus α-helical repeat proteins (48,49) and on deletion studies of LRR proteins (25,50) have shown that the interfacial interactions between repeats are strongly stabilizing. Thus, we expect the highly stable C terminus of PP32 to protect neighboring repeats from exchange, broadening the observed stability map toward the N terminus.…”

The Yersinia Type III secretion effector YopM Is an E3 ubiquitin ligase that induced necrotic cell death by targeting NLRP3

“…27, 28, 29, 30 YopM has a nuclear localization signal in its C-terminus, and is translocated into the nucleus via an endocytic pathway. 31, 32, 33 The first identified targets of YopM were ribosomal S6 protein kinase 1 (RSK1) and protein kinase C-like 2 (PRK2).…”

Comparing protein folding in vitro and in vivo: foldability meets the fitness challenge

“…Moreover, recent work from several groups suggests that, even though large proteins can generally be broken down into smaller units by domain dissection, the folding of these component domains may not be independent, and thus what is seen for free-standing small-domain proteins may not be applicable to the universe of larger proteins in the proteomes of all organisms. Specifically, the domains of repeat proteins have been found to display context-dependent folding [15,16]. In addition, the coupling of domains of large proteins is often a key part of the function of the large protein [17].…”