From Protein Folding to Protein Binding

Proteins are involved in many essential roles in the cell including controlling cell dynamics, mobilizing the intracellular response, cell shape etc. A large population of proteins are required to interact with other organelles or other proteins to accomplish their function. Studying protein folding and stability is a great approach to guide the understanding of protein interactions, functions and structures. To implement Gibbs folding free energy of a protein (∆G_folding), the structural information of the native (folded) and unfolded (denatured) state is needed. In this dissertation, we build the ensemble structures for the unfolded state utilizing a random coil model; which is applied to generate the structures for the intrinsically disordered proteins/regions (IDPs/IDRs) as well. These structures are validated using the experimental pKa values of titratable residues. Several studies have shown that the electrostatic interactions between residues in the unfolded structure causes their pKa values to be perturbed in the denatured state compared to those in the native structure. Furthermore, these ensemble structures of the unfolded state are used to calculate Gibbs folding free energy changes of a protein induced by a point mutation (ΔΔG) by implementing Molecular Mechanics Poisson-Boltzmann (MMPBSA) and machine learning (ML) methods. Comparing our estimations with other available servers with this regard, our approach presents quite well predictions employing only physical parameters based on the Gibbs folding free energy change upon a point mutation. Finally, we study the binding of dynein microtubule binding domain (MTBD) and microtubule (MT), specifically by investigating the role of the IDRs in the C-terminal domains of tubulins (called E-hooks and known to be populated with the acidic residues). We show that the transient or dynamic binding occurs between E-hooks and MTBDs, whereas E-hooks exert electrostatic forces on MTBD to provide a “soft-landing” for the MTBD. Furthermore, we indicate the importance of some key residues of MTBD in binding to the MT through the interaction with E-hooks that may provide the essential information for disease studies linked to the mutations in motor proteins

E-hooks provide guidance and a soft landing for the microtubule binding domain of dynein

Macromolecular binding is a complex process that involves sensing and approaching the binding partner, adopting the proper orientation, and performing the physical binding. We computationally investigated the role of E-hooks, which are intrinsically disordered regions (IDRs) at the C-terminus of tubulin, on dynein microtubule binding domain (MTBD) binding to the microtubule as a function of the distance between the MTBD and its binding site on the microtubule. Our results demonstrated that the contacts between E-hooks and the MTBD are dynamical; multiple negatively charted patches of amino acids on the E-hooks grab and release the same positively charged patches on the MTBD as it approaches the microtubule. Even when the distance between the MTBD and the microtubule was greater than the E-hook length, the E-hooks sensed and guided MTBD via long-range electrostatic interactions in our simulations. Moreover, we found that E-hooks exerted electrostatic forces on the MTBD that were distance dependent; the force pulls the MTBD toward the microtubule at long distances but opposes binding at short distances. This mechanism provides a “soft-landing” for the MTBD as it binds to the microtubule. Finally, our analysis of the conformational states of E-hooks in presence and absence of the MTBD indicates that the binding process is a mixture of the induced-fit and lock-and-key macromolecular binding hypotheses. Overall, this novel binding mechanism is termed “guided-soft-binding” and could have broad-reaching impacts on the understanding of how IDRs dock to structured proteins

Processivity vs. Beating: Comparing Cytoplasmic and Axonemal Dynein Microtubule Binding Domain Association with Microtubule

This study compares the role of electrostatics in the binding process between microtubules and two dynein microtubule-binding domains (MTBDs): cytoplasmic and axonemal. These two dyneins are distinctively different in terms of their functionalities: cytoplasmic dynein is processive, while axonemal dynein is involved in beating. In both cases, the binding requires frequent association/disassociation between the microtubule and MTBD, and involves highly negatively charged microtubules, including non-structured C-terminal domains (E-hooks), and an MTBD interface that is positively charged. This indicates that electrostatics play an important role in the association process. Here, we show that the cytoplasmic MTBD binds electrostatically tighter to microtubules than to the axonemal MTBD, but the axonemal MTBD experiences interactions with microtubule E-hooks at longer distances compared with the cytoplasmic MTBD. This allows the axonemal MTBD to be weakly bound to the microtubule, while at the same time acting onto the microtubule via the flexible E-hooks, even at MTBD–microtubule distances of 45 Å. In part, this is due to the charge distribution of MTBDs: in the cytoplasmic MTBD, the positive charges are concentrated at the binding interface with the microtubule, while in the axonemal MTBD, they are more distributed over the entire structure, allowing E-hooks to interact at longer distances. The dissimilarities of electrostatics in the cases of axonemal and cytoplasmic MTBDs were found not to result in a difference in conformational dynamics on MTBDs, while causing differences in the conformational states of E-hooks. The E-hooks’ conformations in the presence of the axonemal MTBD were less restricted than in the presence of the cytoplasmic MTBD. In parallel with the differences, the common effect was found that the structural fluctuations of MTBDs decrease as either the number of contacts with E-hooks increases or the distance to the microtubule decreases

Gaussian-Based Smooth Dielectric Function: A Surface-Free Approach for Modeling Macromolecular Binding in Solvents

Conventional modeling techniques to model macromolecular solvation and its effect on binding in the framework of Poisson-Boltzmann based implicit solvent models make use of a geometrically defined surface to depict the separation of macromolecular interior (low dielectric constant) from the solvent phase (high dielectric constant). Though this simplification saves time and computational resources without significantly compromising the accuracy of free energy calculations, it bypasses some of the key physio-chemical properties of the solute-solvent interface, e.g., the altered flexibility of water molecules and that of side chains at the interface, which results in dielectric properties different from both bulk water and macromolecular interior, respectively. Here we present a Gaussian-based smooth dielectric model, an inhomogeneous dielectric distribution model that mimics the effect of macromolecular flexibility and captures the altered properties of surface bound water molecules. Thus, the model delivers a smooth transition of dielectric properties from the macromolecular interior to the solvent phase, eliminating any unphysical surface separating the two phases. Using various examples of macromolecular binding, we demonstrate its utility and illustrate the comparison with the conventional 2-dielectric model. We also showcase some additional abilities of this model, viz. to account for the effect of electrolytes in the solution and to render the distribution profile of water across a lipid membrane