Conformational and Dynamic Motifs in the Biomolecular Recognition of Glycan and Acetyl Functionalities

Abstract

Biomolecular recognition is a fundamental principle underlying all cellular processes, driven by specific and tightly regulated interactions. This thesis explores the structural basis of recognition mechanisms in two distinct contexts: protein-glycan interactions and post-translational modification, specifically lysine acetylation as a regulator of recognition. The work is presented in two parts. In Part I, the focus is on a non-mitogenic lectin, horcolin, and its recognition of mannose. Lectins are sugar-binding proteins that have shown considerable promise as antiviral agents because of their ability to interact with envelope glycoproteins present on the surface of viruses such as HIV-1. However, their therapeutic potential has been compromised by their mitogenicity that stimulates uncontrolled division of T-lymphocytes. Horcolin, a member of the jacalin family of lectins, tightly binds the HIV-1 envelope glycoprotein gp120 and neutralizes HIV-1 particles, but is non-mitogenic. In this thesis, we combine X-ray crystallography and NMR spectroscopy to obtain atomic resolution insights into the structure of horcolin and the molecular basis for its carbohydrate recognition. Each protomer of the horcolin dimer adopts a canonical β-prism I fold with three Greek key motifs and carries two carbohydrate binding sites. The carbohydrate molecule binds in a negatively charged pocket and is stabilized by backbone and side chain hydrogen bonds to conserved residues in the ligand binding loop. NMR titrations reveal a two-site binding mode and equilibrium dissociation constants for the two binding sites determined from 2D lineshape modeling are 4-fold different. Single-binding-site variants of horcolin confirm the dichotomy in binding sites and suggest that there is allosteric communication between the two sites. An analysis of the horcolin structure shows a network of hydrogen bonds linking the two carbohydrate binding sites directly and through a secondary binding site, and this coupling between the two sites is expected to assume importance in the interaction of horcolin with high-mannose glycans found on viral envelope glycoproteins.

Additionally, we also explored the conformational frustration that exists in protein-glycan interfaces using horcolin as our model system. Saturation transfer and relaxation dispersion NMR experiments reveal that the lectin-glycan interface is conformationally frustrated, resulting in the formation of an excited state with a millisecond timescale lifetime. There is a rearrangement of the quaternary structure of horcolin in this excited state that manifests as a non-canonical tetramer. The glycan binding site is sequestered at the tetrameric interface, suggesting that the tetramer could serve as an autoinhibitory conformation. However, glycan recognition itself occurs via the major dimeric conformation through a ‘ground-state conformational selection’ mechanism. We also demonstrate that the tetramer is destabilized by mannose and that conformational frustration is alleviated in the lectin-glycan complex. Our work illustrates how the architecture of biomolecular assemblies is moulded in response to conflicting evolutionary signals such as folding and recognition. The work also provides insights into protein-glycan recognition that could have potential implications for deploying lectins as antiviral agents. In Part II, we investigated the role of lysine acetylation in the mammalian circadian clock protein BMAL1. The circadian clock is operated by an auto-regulated transcription-translation feedback loop, finely regulated by several post-translational modifications. Studies have shown the role of acetylation of K537 of BMAL1 in initiating the negative limb of the circadian cycle, where the intrinsically disordered C-terminus of BMAL1 binds to its cognate partner CRY1 and sustains the rhythmicity of the cycle. We sought to understand the structural basis for regulation through acetylation. Secondary structure propensities from NMR chemical shifts indicate the presence of transient helicity in the transactivation domain. Furthermore, resonance changes upon acetylation are restricted to residues near the acetylation site, indicating that acetylation does not modify the overall conformation. Fast backbone dynamics in the picosecond-nanosecond timescale probed through measuring R1, R2 and heteronuclear NOE reveal that subtle changes occur in the flexibility of the backbone upon acetylation. Additionally, we developed an in vitro enzymatic approach to acetylate the conserved K537 residue. Using NMR spectroscopy, we provide a simple way to detect acetyl moieties at the sidechain of lysine residues. Significant changes in the resonances of acetyl-lysine as compared to the unmodified lysine allow a direct identification of acetylation. Using multiple bond coherence transfer from the sidechain amide of the acetyl-lysine to other sidechain atoms, we establish a method to also estimate acetylation efficiency. This lays the groundwork for future studies on the conformational and dynamic consequences of acetylation in circadian regulation. Together, the findings presented in this thesis deepen our understanding of the structural and dynamic principles underlying biomolecular recognition and regulation in complex biological systems.