Abstract
Proteins are subject to spontaneous rearrangements of their backbones. Most prominently, asparagine and aspartate residues isomerize to their β-linked isomer, isoaspartate (isoAsp), on time scales ranging from days to centuries. Such modifications are typically considered "molecular wear-and-tear", destroying protein function. However, the observation that some proteins, including the essential bacterial enzyme MurA, harbor stoichiometric amounts of isoAsp suggests that this modification can confer advantageous properties. Here, we demonstrate that nature exploits an isoAsp residue within a hairpin to stabilize MurA. We found that isoAsp formation in MurA is unusually rapid and critically dependent on folding status. Moreover, perturbation of the isoAsp-containing hairpin via site-directed mutagenesis causes aggregation of MurA variants. Structural mass spectrometry revealed that this effect is caused by local protein unfolding in MurA mutants. Our findings demonstrate that MurA evolved to "mature" via a spontaneous post-translational incorporation of a β-amino acid, which raises the possibility that isoAsp-containing hairpins may serve as a structural motif of biological importance.
Highlights
Proteins are subject to spontaneous rearrangements of their backbones
To verify the presence and extent of the isoAsp modification in E. cloacae MurA, we produced the enzyme in Escherichia coli as an N-terminal fusion with a hexahistidine
As judged by SDS PAGE (Figure S5), and the identity of the protein was confirmed by mass spectrometry (Figure S6a)
Summary
Asparagine and aspartate residues isomerize to their β-linked isomer, isoaspartate (isoAsp), on time scales ranging from days to centuries Such modifications are typically considered “molecular wear-and-tear”, destroying protein function. Over typical protein lifetimes, asparagine (Asn) and aspartate (Asp) residues can spontaneously isomerize into isoaspartate (isoAsp) featuring an unusual β-peptidic linkage (Figure 1a).[1−3] This process is initiated by the nucleophilic attack of the (deprotonated) backbone amide nitrogen on the side chain amide or acid group in the case of Asn and Asp, respectively. The resulting succinimide intermediate hydrolyses with a slight preference (70−85%) in the α position, yielding a mixture of isoAsp and Asp residues.[1] In model peptides, this process occurs on time scales ranging from days to years, with Asn−Gly sequences rearranging most rapidly.[4] The rates are typically lower in proteins, especially for Asn residues that are embedded in secondary structure motifs or otherwise engaged in H-bonds involving the N−H of the i+1 residue.[5]. In other structurally characterized MurA homologues, more residues are needed to provide a similar structure. β-Peptidic linkages have been shown to provide unique structural features, primarily in the context of foldamers.[16,17]
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