Metal Cation Coupled Multiscale Conformational Motions Of Protein

Metalloprotein is a term for a protein that is characterized by the interaction with one or more metal ion cofactors to stabilize the fold and/or to make them functional. It is estimated that more than one third of all proteins require metals to carry out their functions, ranging from storage and transport of proteins to signal transduction. Understanding the molecular basis of metal binding and the subsequent functional conformational changes in metalloproteins is of fundamental importance. Despite the accumu-lating knowledge from experiment and theory on biochemical properties of metalloproteins, the molecular mechanism and physical principles governing the metal-coupled multi-scale conformational motions of metalloproteins remain elusive. Here in this dissertation, I present three of our works focusing on the water-mediated coordination in Ca2+ions induced asymmetric allosteric transitions in the N-terminal do-main of Calmodulin, and the localized frustration and binding-induced conformational change in recog-nition of RNA molecules by multi-domain zinc-finger proteins.Calcium (Ca2+) binding to the helix-loop-helix motifs (termed EF-hands) in calmodulin (CaM) leads to large conformational changes, resulting in the exposure of the hydrophobic cores that are recognition sites for a number of target proteins involved in a variety of cell signaling events. Despite the physio-logical importance, the mechanism of Ca2+-mediated allosteric transitions in CaM is not understood at the molecular level. We use all-atom MD simulations in conjunction with metadynamics to investigate the coupling between the Ca2+binding and the conformational change of the N-terminal domain of CaM (nCaM) containing two EF-hands (EF1and EF2hands). We show that despite being structurally similar, there is an asymmetry in the structural response in the two EF-hands upon Ca2+binding. Although the N-terminus EF1hand can adopt Ca2+-triggered open conformation independently, the stability of the open state of the EF2hand requires allosteric communication with the EF1hand. We show that the exposure of hydrophobic sites required for recognition by target proteins to initiate signal transduction occurs by cal-cium induced rotation of the helices of EF-hands with the hydrophobic core serving as the pivot. In both EF-hands chelation to the oxygen atoms of the charged residues in the loop requires partial dehydration of Ca2+. Our work also reveals a water-bridged coordination mechanism of the Ca2+binding in which the bridging water molecules reduce the entropy penalty during the dehydration of Ca2+, thus contributing to the efficient ligand binding and allosteric motions of the nCaM. The atomically detailed picture for the allosteric transitions of the two EF-hands in nCaM, which are the first events in mediating a variety of intracellular processes, reveal the complex interplay between the discrete water molecules, dehydration of Ca2+, and nCaM structural changes.Protein TFIIIA is composed of nine tandemly arranged Cys2His2zinc fingers. It can bind either to the5S rRNA gene as a transcription factor or to the5S rRNA transcript as a chaperone. Although structural and biochemical data provided valuable information on the recognition between the TFIIIA and the5S DNA/RNA, the involved conformational motions and energetic factors contributing to the binding affinity and specificity remain unclear. We conducted MD simulations and MM/GBSA (Molecular Mechanics/Generalized Born Surface Area) calculations to investigate the binding-induced conformational changes in the recognition of the5S rRNA by the central three zinc-fingers of TFIIIA and the energetic factors that influence the binding affinity and specificity at an atomistic level. Our results revealed drastic inter-domain conformational changes between these three zinc-fingers, involving the exposure/embedding of several crucial DNA/RNA binding residues, which can be related to the competition between DNA and RNA for the binding of TFIIIA. We also showed that the specific recognition between the finger4/finger6and the5S rRNA introduces frustrations to the non-specific interactions between the finger5and the5S rRNA, which may be important to achieve optimal binding affinity and specificity.Protein Lin28recognizes pre-let-7miRNAs through direct interactions between its zinc-knuckle type zinc-finger (ZnF) domains and the terminal loop of pre-let-7, resulting in the inhibition of the synthesis of mature let-7miRNAs. The Lin28-pre-let-7interaction is particularly notable because of its role in the tumorigenesis. However, the involved conformational changes and the crucial physical interactions that affect the sequence-specific recognition remain unclear. We conducted MD simulations in conjunction with MM/GBSA and energy decomposition calculations to investigate the miRNA binding-induced con-formational changes of the zinc-fingers and the residual level amino acid-nucleic acid interactions that influence the binding specificity. We showed that the binding of the miRNA results in the inter-domain conformational changes of the two zinc-finger domains, including changes of the spatial relationships between several nucleobase-binding amino acids. We also observed mutation-induced weakening of the affinity of the Lin28-pre-let-7binding, which reveals the importance of the stacking interactions between the side-chains of Tyr140, His148, His162and the bases of nucleic acid G2and G5in the specific recog-nition of pre-let-7by the ZnFs of Lin28.This dissertation brings forth several innovations:i) We completely constructed the free energy landscape of the Ca2+binding coupled conformational changes of nCaM, and revealed the binding order of the Ca2+to the native ligands, and the consequent rotation of EF-hand helices, with the hydrophobic core serving as a pivot, ii) Using metadynamics and all-atom MD simulations, we revealed a water-bridged coordination mechanism of metal ions, in which the bridging water molecule reduces the entropy penalty during the dehydration process of metal ion, thus contributing to the efficient ligand binding and allosteric motions of proteins. iii) We demonstrated the conformational changes of the multi-domain protein TFIIIA during the binding with RNA. These inter-domain conformational changes involve the exposure/embedding of several crucial nucleic acid binding sites. This mechanism may contribute to the auto-regulation in the synthesis of5S rRNA. iv)"Local frustration" is a reside-level concept derived from the protein folding field. Here we revealed the "local frustration" on the protein domain level during the binding of biomolecules:the sequence-specific interactions of some domain can result in the weakening of non-specific interactions of other parts in the same complex. This is important for the balance between these two binding modes and for achieving optimal binding afffnity.This dissertation is organized as the following:· Chapter Ⅰ is a general introduction to the background of our research topics, as well as the methods and techniques we used in our work.· In Chapter Ⅱ, we discussed the Ca2+binding induced conformational changes of nCaM, and the crucial roles water molecules play in the binding process.· In Chapter Ⅲ, we studied the conformational changes during the binding of TFIIIA and5S rRNA, and the energy factors that contribute to the binding affinity and specificity.· In Chapter Ⅳ, we demonstrated the recognition mechanism of pre-let-7miRNA by the two zinc-finger domains in Lin28, and the importance of amino acid side-chain-nucleic acid base stacking interactions in the sequence-specific interactions.· Chapter Ⅴ is a summary of this dissertation.