Characterizing the link between dynamics and function in proteins that violate the structure-function paradigm

Abstract

The structure-function paradigm states that the three-dimensional structure of a protein is dictated by its amino acid sequence, which influences its function. However, this paradigm fails to explain the behaviour of proteins that can sample multiple conformations, particularly intrinsically disordered proteins (IDPs) and metamorphic proteins. IDPs lack a stable three dimensional structure and can adopt multiple conformations to perform distinct functions, while metamorphic proteins can switch between different structures to perform different functions. Characterizing the free energy landscape of IDPs and metamorphic proteins is crucial for understanding their function, as it can reveal minor conformations that contribute to their functioning. However, studying these conformations can be challenging due to their relatively low populations and short lifetimes. We have examined two proteins that violate the sequence structure-function paradigm: an intrinsically disordered protein (IDP) CytRN and a metamorphic protein KaiB, and extensively characterised minor conformations present in both proteins, thus revealing their crucial contributions to function. In this thesis, we have studied the intrinsically disordered DNA binding domain (CytRN) of the cytidine repressor (CytR), a LacR family transcriptional repressor found in E.coli. Through conventional three-dimensional NMR experiments, we have assigned the protein backbone, and using Chemical Exchange Saturation Transfer (CEST) NMR experiments, we discover that CytRN coexists in equilibrium with a minor conformation/excited state. We recorded multinuclear CEST on backbone nuclei (15N, 1HN, 13C’ and 13C⍺) to obtain the chemical shifts of this excited state. To derive additional restraints for solving the structure of CytRN excited state, we have measured residual dipolar couplings (RDC) using CEST experiments in alignment media. We obtained RDCs in two different alignment media, the lyotropic phase formed by a PEG/octanol mixture and stretched polyacrylamide gels, using 15N CEST and 1HN CEST-based approaches. Subsequently, employing RDCs and chemical shifts obtained from multinuclear CEST experiments as restraints, we calculated the excited state structure using CS-Rosetta and find that the CytRN excited state is globally folded. The excited state structure adopts a three-helix bundle conformation similar to the DNA-bound form of CytRN. However, we observe minor 10 differences between the excited state and the DNA-bound form, such as variations in the length of the recognition helix and orientations in sidechains near the DNA binding site. Next, we aimed to elucidate the mechanism by which CytRN binds DNA. To pursue this aim, we have employed the Double Resonance DANTE-CEST (DRD-CEST) experiment, which was developed recently and is based on DANTE-CEST. Our findings reveal that the excited state is selectively chosen to bind DNA through a conformational selection mechanism and this pathway is about 100 times more likely to occur than the induced fit pathway. Overall, our study has demonstrated that the CytRN IDP transiently samples a fully folded conformation, and this excited state is very similar to the DNA-bound form. Mechanistic studies reveal that this excited state regulates the function of intrinsically disordered CytRN by being the state that binds DNA. In the second part of this thesis, we present our findings on the metamorphic protein KaiB which is a constituent of the core circadian oscillator in cyanobacteria. As a metamorphic protein, KaiB adopts two conformations, referred to as the ground state and the fold-switched state, with similar free energies. Our objective is to characterize the free energy landscape of native KaiB and to identify intermediates that may represent the starting points for the interconversion of KaiB from the ground state to the fold-switched state. We performed CEST experiments on a dimeric KaiB construct and the 15N CEST experiments reveal that KaiB samples a thermally accessible minor conformation. We find that the residues undergoing exchange are located at the dimer interface, suggesting that the excited state sampled by KaiB is a monomer. Concentration-based CEST experiments further support this observation that the excited state is indeed a monomer. Using multinuclear CEST experiments, we obtained the chemical shifts of all backbone nuclei in the excited state. The chemical shifts of KaiB at the sites of exchange do not correlate well with those of the disordered or the foldswitched states, suggesting that the structure of this excited state neither resembles the disordered nor the fold-switched state completely. Structure calculation of the excited state using CS-Rosetta reveals that the excited state structure comprises of secondary structural elements from both the ground state and the foldswitched state. This suggests that the excited state could possibly represent an on-pathway intermediate in the fold-switching pathway of KaiB. Ongoing studies aim to validate the structure by using structure-guided mutagenesis to stabilize or destabilize the excited state.