Distinct roles of the major binding residues in the cation-binding pocket of the melibiose transporter MelB.

Salmonella enterica serovar Typhimurium melibiose permease (MelBSt) is a prototype of the major facilitator superfamily (MFS) transporters, which play important roles in human health and diseases. MelBSt catalyzed the symport of galactosides with Na+, Li+, or H+ but prefers the coupling with Na+. Previously, we determined the structures of the inward- and outward-facing conformation of MelBSt and the molecular recognition for galactoside and Na+. However, the molecular mechanisms for H+- and Na+-coupled symport remain poorly understood. In this study, we solved two x-ray crystal structures of MelBSt, the cation-binding site mutants D59C at an unliganded apo-state and D55C at a ligand-bound state, and both structures display the outward-facing conformations virtually identical as published. We determined the energetic contributions of three major Na+-binding residues for the selection of Na+ and H+ by free energy simulations. Transport assays showed that the D55C mutant converted MelBSt to a solely H+-coupled symporter, and together with the free-energy perturbation calculation, Asp59 is affirmed to be the sole protonation site of MelBSt. Unexpectedly, the H+-coupled melibiose transport exhibited poor activities at greater bulky ΔpH and better activities at reversal ΔpH, supporting the novel theory of transmembrane-electrostatically localized protons and the associated membrane potential as the primary driving force for the H+-coupled symport mediated by MelBSt. This integrated study of crystal structure, bioenergetics, and free energy simulations, demonstrated the distinct roles of the major binding residues in the cation-binding pocket of MelBSt.

Distinct roles of the major binding residues in the cation-binding pocket of MelB.

Salmonella enterica serovar Typhimurium melibiose permease (MelBSt) is a prototype of the major facilitator superfamily (MFS) transporters, which play important roles in human health and diseases. MelBSt catalyzed the symport of galactosides with either H+, Li+, or Na+, but prefers the coupling with Na+. Previously, we determined the structures of the inward- and outward-facing conformation of MelBSt, as well as the molecular recognition for galactoside and Na+. However, the molecular mechanisms for H+- and Na+-coupled symport still remain poorly understood. We have solved two x-ray crystal structures of MelBSt cation-binding site mutants D59C at an unliganded apo-state and D55C at a ligand-bound state, and both structures display the outward-facing conformations virtually identical as published previously. We determined the energetic contributions of three major Na+-binding residues in cation selectivity for Na+ and H+ by the free energy simulations. The D55C mutant converted MelBSt to a solely H+-coupled symporter, and together with the free-energy perturbation calculation, Asp59 is affirmed to be the sole protonation site of MelBSt. Unexpectedly, the H+-coupled melibiose transport with poor activities at higher ΔpH and better activities at reversal ΔpH was observed, supporting that the membrane potential is the primary driving force for the H+-coupled symport mediated by MelBSt. This integrated study of crystal structure, bioenergetics, and free energy simulations, demonstrated the distinct roles of the major binding residues in the cation-binding pocket.

Mobile barrier mechanisms for Na+-coupled symport in an MFS sugar transporter.

While many 3D structures of cation-coupled transporters have been determined, the mechanistic details governing the obligatory coupling and functional regulations still remain elusive. The bacterial melibiose transporter (MelB) is a prototype of major facilitator superfamily transporters. With a conformation-selective nanobody, we determined a low-sugar affinity inward-facing Na+-bound cryoEM structure. The available outward-facing sugar-bound structures showed that the N- and C-terminal residues of the inner barrier contribute to the sugar selectivity. The inward-open conformation shows that the sugar selectivity pocket is also broken when the inner barrier is broken. Isothermal titration calorimetry measurements revealed that this inward-facing conformation trapped by this nanobody exhibited a greatly decreased sugar-binding affinity, suggesting the mechanisms for substrate intracellular release and accumulation. While the inner/outer barrier shift directly regulates the sugar-binding affinity, it has little or no effect on the cation binding, which is supported by molecular dynamics simulations. Furthermore, the hydron/deuterium exchange mass spectrometry analyses allowed us to identify dynamic regions; some regions are involved in the functionally important inner barrier-specific salt-bridge network, which indicates their critical roles in the barrier switching mechanisms for transport. These complementary results provided structural and dynamic insights into the mobile barrier mechanism for cation-coupled symport.

Mobile barrier mechanisms for Na+-coupled symport in an MFS sugar transporter.

While many 3D structures of cation-coupled transporters have been determined, the mechanistic details governing the obligatory coupling and functional regulations still remain elusive. The bacterial melibiose transporter (MelB) is a prototype of the Na+-coupled major facilitator superfamily transporters. With a conformational nanobody (Nb), we determined a low-sugar affinity inward-facing Na+-bound cryoEM structure. Collectively with the available outward-facing sugar-bound structures, both the outer and inner barriers were localized. The N- and C-terminal residues of the inner barrier contribute to the sugar selectivity pocket. When the inner barrier is broken as shown in the inward-open conformation, the sugar selectivity pocket is also broken. The binding assays by isothermal titration calorimetry revealed that this inward-facing conformation trapped by the conformation-selective Nb exhibited a greatly decreased sugar-binding affinity, suggesting the mechanisms for the substrate intracellular release and accumulation. While the inner/outer barrier shift directly regulates the sugar-binding affinity, it has little or no effect on the cation binding, which is also supported by molecular dynamics simulations. Furthermore, the use of this Nb in combination with the hydron/deuterium exchange mass spectrometry allowed us to identify dynamic regions; some regions are involved in the functionally important inner barrier-specific salt-bridge network, which indicates their critical roles in the barrier switching mechanisms for transport. These complementary results provided structural and dynamic insights into the mobile barrier mechanism for cation-coupled symport.

On the Simulation of Thermal Isomerization of Molecular Photoswitches in Biological Systems.

Molecular photoswitches offer precise, reversible photocontrol over biomolecular functions and are promising light-regulated drug candidates with minimal side effects. Quantifying thermal isomerization rates of photoswitches in their target biomolecules is essential for fine-tuning their light-controlled drug activity. However, the effects of protein binding on isomerization kinetics remain poorly understood, and simulations are crucial for filling this gap. Challenges in the simulation include describing multireference electronic structures near transition states, disentangling competing reaction pathways, and sampling protein-ligand interactions. To overcome these challenges, we used multiscale simulations to characterize the thermal isomerization of photostatins (PSTs), which are light-regulated microtubule inhibitors for potential cancer phototherapy. We employed a new ab initio multireference electronic structure method in a quantum mechanics/molecular mechanics setting and combined it with enhanced sampling techniques to characterize the cis to trans free-energy profiles of three PSTs in a vacuum, aqueous solution, and tubulin dimer. The significant advantage of our novel approach is the efficient treatment of the multireference character in PSTs' electronic wavefunction throughout the conformational sampling of protein-ligand interactions along their isomerization pathways. We also benchmarked our calculations using high-level ab initio multireference electronic structure methods and explored the competing isomerization pathways. Notably, calculations in a vacuum and implicit solvent models cannot predict the order of the PSTs' thermal half-lives in the aqueous solution observed in the experiment. Only by explicitly treating the solvent molecules can the correct order of isomerization kinetics be reproduced. Protein binding perturbs free-energy barriers due to hydrogen bonding between PSTs and nearby polar residues. Our work generates comprehensive, high-quality benchmark data and offers guidance for selecting computational methods to study the thermal isomerization of photoswitches. Ab initio multireference free-energy calculations in explicit molecular environments are crucial for predicting the effects of substituents on the thermal half-lives of photoswitches in biological systems.

Multiscale simulation unravels the light-regulated reversible inhibition of dihydrofolate reductase by phototrexate.

Molecular photoswitches are widely used in photopharmacology, where the biomolecular functions are photo-controlled reversibly with high spatiotemporal precision. Despite the success of this field, it remains elusive how the protein environment modulates the photochemical properties of photoswitches. Understanding this fundamental question is critical for designing more effective light-regulated drugs with mitigated side effects. In our recent work, we employed first-principles non-adiabatic dynamics simulations to probe the effects of protein on the trans to cis photoisomerization of phototrexate (PTX), a photochromic analog of the anticancer therapeutic methotrexate that inhibits the target enzyme dihydrofolate reductase (DHFR). Building upon this study, in this work, we employ multiscale simulations to unravel the full photocycle underlying the light-regulated reversible inhibition of DHFR by PTX, which remains elusive until now. First-principles non-adiabatic dynamics simulations reveal that the cis to trans photoisomerization quantum yield is hindered in the protein due to backward isomerization on the ground-state following non-adiabatic transition, which arises from the favorable binding of the cis isomer with the protein. However, free energy simulations indicate that cis to trans photoisomerization significantly decreases the binding affinity of the PTX. Thus, the cis to trans photoisomerization most likely precedes the ligand unbinding from the protein. We propose the most probable photocycle of the PTX-DHFR system. Our comprehensive simulations highlight the trade-offs among the binding affinity, photoisomerization quantum yield, and the thermal stability of the ligand's different isomeric forms. As such, our work reveals new design principles of light-regulated drugs in photopharmacology.

Effects of Enzyme-Ligand Interactions on the Photoisomerization of a Light-Regulated Chemotherapeutic Drug.

Molecular photoswitches permit using light to control protein activity with high spatiotemporal resolutions, thereby alleviating the side effects of conventional chemotherapy. However, due to the challenges in probing ultrafast photoisomerization reactions in biological environments, it remains elusive how the protein influences the photochemistry of the photoswitches, which hampers the rational design of light-regulated therapeutics. To overcome this challenge, we employed first-principles nonadiabatic dynamics simulations to characterize the photodynamics of the phototrexate (PTX), a recently developed photoswitchable anticancer chemotherapeutic that reversibly inhibits its target enzyme dihydrofolate reductase (DHFR). Our simulations show that the protein environment impedes the trans to cis photoisomerization of the PTX. The confinement in the ligand-binding cavity slows down the isomerization kinetics and quantum yield of the photoswitch by reshaping its conical intersection, increasing its excited-state free-energy barrier and quenching its local density fluctuations. Also, the protein environment results in a suboptimal binding mode of the photoproduct that needs to undergo large structural rearrangement to effectively inhibit the enzyme. Therefore, we predict that the PTX's trans → cis photoisomerization in solution precedes its binding with the protein, despite the favorable binding energy of the trans isomer. Our findings highlight the importance of the protein environment on the photochemical reactions of the molecular photoswitches. As such, our work represents an important step toward the rational design of light-regulated drugs in photopharmacology.

Protein confinement fine-tunes aggregation-induced emission in human serum albumin.

Luminogens exhibiting aggregation-induced-emission characteristics (AIEgens) have been designed as sensitive biosensors thanks to their "turn-on" fluorescence upon target binding. However, their AIE mechanism in biomolecules remains elusive except for the qualitative picture of restricted intramolecular motions. In this work, we employed ab initio simulations to investigate the AIE mechanism of two tetraphenylethylene derivatives recently developed for sensitive detection of human serum albumin (HSA) in biological fluids. For the first time, we quantified the ab initio free energy surfaces and kinetics of AIEgens to access the conical intersections on the excited state in the protein and aqueous solution, using a novel first-principles electronic structure method that incorporates both static and dynamic electron correlations. Our simulations accurately reproduce the experimental spectra and high-level correlated electronic structure calculations. We found that in HSA the internal conversion through the cyclization reaction is preferred over the isomerization around the central ethylenic double bond, whereas in the aqueous solution the reverse is true. Accordingly, the protein environment is able to moderately speed up certain non-radiative decay pathways, a new finding that is beyond the prediction of the existing model of restricted access to a conical intersection (RACI). As such, our findings highlight the complicated effects of the protein confinement on the competing non-radiative decay channels, which has been largely ignored so far, and extend the existing theories of AIE to biological systems. The new insights and the multiscale computational methods used in this work will aid the design of sensitive AIEgens for bioimaging and disease diagnosis.

A bioprinted composite hydrogel with controlled shear stress on cells.

In recent decades, three dimensional (3D) bio-printing technology has found widespread use in tissue engineering applications. The aim of this study is to scrutinize different parameters of the bioprinter - with the help of simulation software - to print a hydrogel so much so that avoid high amounts of shear stress which is detrimental for cell viability and cell proliferation. Rheology analysis was done on several hydrogels composed of different percentages of components: alginate, collagen, and gelatin. The results have led to the combination of percentages collagen:alginate:gelatin (1:4:8)% as the best condition which makes sol-gel transition at room temperature possible. The results have shown the highest diffusion rate and cell viability for the cross-linked sample with 1.5% CaCl2 for the duration of 1 h. Finally, we have succeeded in printing the hydrogel that is mechanically strong with suitable degradation rate and cell viability.