Structural and Functional Consequences of a Protein Backbone Modification

Student thesis: Doctoral ThesisDoctor of Philosophy

Abstract

Over the lifetime of proteins, asparagine (Asn) and aspartate (Asp) residues can spontaneously isomerize into isoAspartate (isoAsp) which features an unusual β-peptidic linkage. IsoAsp formation is typically considered as protein damage and associated with several pathologies. To counteract negative effects of isoAsp formation, Nature has evolved a ‘repair’ enzyme, protein carboxyl methyltransferase (PIMT), to partially restore isoAsp back to Asp. My research aims to overthrow this paradigm by investigating gain-of-function maturation processes involving isoAsp formation and the potential for isoAsp formation as a biochemical signal.

The bacterial enzyme MurA features an unusually tight hairpin involving an isoAsp residue, which is formed post-translationally from Asn67. In the first part of my thesis I examined the function of this unusual motif. My experiments show that the fast and stoichiometric isoAsp formation in MurA is not subjected to PIMT repair under native conditions. Moreover, I found that isoAsp formation in folded MurA is >10 times faster than in the corresponding unfolded peptide. Mutations of Asn67 and its surrounding residues cause protein aggregation, slightly decreased protein activity and reduced isoAsp formation kinetics. To understand the aggregation and protein stability, we performed chemical and thermal denaturation and HDX-MS experiments. The results show that isoAsp increases protein stability by rigidifying its surrounding regions. Altering the unique connectivity of isoAsp through an Asp mutation increases local unfolding of the hairpin which propagates to neighbouring structural elements on a timescale of minutes to hours. Collectively, these results demonstrate that isoAsp can be beneficial, and that MurA has evolved to promote its formation. We speculated that isoAsp-containing hairpins might be a recurring structural motif for improving protein stability. To explore this possibility, we characterised the Invasin IpaD which harbours a similar motif; and also attempted to transplant the isoAsp-containing hairpin from MurA to its homolog AroA. However, we found that at least among these examples, MurA is unique in its ability to accumulate the formation of isoAsp.

I also examined the biophysical mechanism of chromatin regulation via isoAsp formation. H4D24me, installed by isoAsp repair methyltransferase PIMT, has been found to be ubiquitously present in different mammalian cell lines, indicating that Asp24 of histone H4 can become spontaneously converted to isoAsp over time. We hypothesise that isoAsp24, the product of histone H4 aging, will initiate its own repair by forcing chromatin into an open conformation by reducing interactions between the H4 N-terminal tail and the acidic patch of neighbouring nucleosomes. Also, isoAsp changes the spacing and direction of the H4 tail which could impact the activity of the H4K20 methyltransferases Set8/Suv4-20. Moreover, chromatin remodeling factors which require binding with the H4 tail potentially also be altered in its activity upon changing Asp24H4 to isoAsp. I employed protein semi-synthesis via SPPS and native chemical ligation to prepare isoAsp24-containing H4 and assembled into designer chromatin arrays to enable direct experimental testing of these possibilities.

In summary of the results, isoAsp formation can be useful for protein properties and its effects may be subtle and as expected, very context-dependent.
Date of Award1 Sept 2022
Original languageEnglish
Awarding Institution
  • King's College London
SupervisorManuel Muller (Supervisor) & Rivka Isaacson (Supervisor)

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