A structural approach to protein intrinsic versatility : the tale of succinyllysine reader GAS41 and palmitoyllysine eraser SIRT3

Abstract

Proteins are sophisticated biomolecules with designs refined over the course of evolution. Many proteins are intrinsically versatile, capable of performing a variety of functions subjecting to the environment and need of the cell. Their versatility is built into sequences and structural folds of each protein but is not easily understood when our hypotheses often overlook them and experiments do not meet the specific working conditions. This thesis argues that in modern molecular biology with a wealth of structural data, computational power and high-throughput experiments, there is a need to take advantage of these resources and explore what is not directly detectable. This study investigates the potential of structural biology at understanding chemical and structural versatility in the context of post-translational modification (PTM) of proteins. Different types of PTMs form complex signalling systems which are an essential part of molecular cell biology. The diversity of PTMs emphasises the need for a complementary population of versatile proteins to regulate the system and propagate information.

Two cases of a PTM reader and a PTM eraser were studied. The first case describes the pH-dependent recognition of succinyllysine by GAS41 YEATS domain and discusses the possible contexts for the functional relevance of its chemical versatility. Succinylation at H3K122 was identified to be a ligand for GAS41 and its yeast homologue Yaf9 by peptide microarray and pull-down assays. The structure of Yaf9 YEATS domain complexed with succinylated H3117-127K122 was solved as well as GAS41 YEATS domain alone. Structural analysis suggests a conserved binding mechanism. A higher binding affinity for succinylated H3117-127K122 at lower pH as measured by surface plasmon resonance was observed. The mutation of a histidine into phenylalanine in the pocket reduced the binding affinity. Cellular assays showed that this succinylated H3K122 readout by GAS41 is associated with its occupancy on the p21 promoter. The second case presents an extensive structural analysis of SIRT3 depalmitoylase activity and discusses how this enzyme is designed for a wide range of PTM substrate lengths. Crystal structures of three different SIRT3 depalmitoylation stages were solved by X-ray crystallography. The superpositions of these structures together with previously reported ones showed unique conformational changes of both the substrate and the enzyme. The palmitoyl group is pushed inwards by the incoming NAD+ and the cofactor-binding loop, which induces SIRT3 sub-domain “breathing” movements and internal side-chain repositioning to extend the pocket. Several residues unique to individual sirtuins that possibly contribute to differential depalmitoylation efficiencies were suggested or reported for mutation experiments. The hinges of the movements were proposed as additional mutation targets. These results are knowledge of the understudied parts of the proteins that can be translated to designing engineered proteins as study tools and aid therapeutic agent development. This provides a motivation for further exploration of conditional functions of biomolecules and promotes the role of structural biology in studying protein versatility.