PROTAC-induced Protein Functional Dynamics in Targeted Protein Degradation.

PROteolysis TArgeting Chimeras (PROTACs) are small molecules that induce target protein degradation via the ubiquitin-proteasome system. PROTACs recruit the target protein and E3 ligase; a critical first step is forming a ternary complex. However, while the formation a ternary complex is crucial, it may not always guarantee successful protein degradation. The dynamics of the PROTAC-induced degradation complex play a key role in ubiquitination and subsequent degradation. In this study, we computationally modelled protein complex structures and dynamics associated with a series of PROTACs featuring different linkers to investigate why these PROTACs, all of which formed ternary complexes with Cereblon (CRBN) E3 ligase and the target protein bromodomain-containing protein 4 (BRD4 BD1 ), exhibited varying degrees of degradation potency. We constructed the degradation machinery complexes with Culling-Ring Ligase 4A (CRL4A) E3 ligase scaffolds. Through atomistic molecular dynamics simulations, we illustrated how PROTAC-dependent protein dynamics facilitate the arrangement of surface lysine residues of BRD4 BD1 into the catalytic pocket of E2/ubiquitin for ubiquitination. Despite featuring identical warheads in this PROTAC series, the linkers were found to affect the residue-interaction networks, and thus governing the essential motions of the entire degradation machine for ubiquitination. These findings offer a dynamic perspective on ligand-induced protein degradation, providing insights to guide future PROTAC design endeavors.

Selective recognition and discrimination of single isomeric changes in peptide strands with a host : guest sensing array.

An indirect competitive binding mechanism can be exploited to allow a combination of cationic fluorophores and water-soluble synthetic receptors to selectively recognize and discriminate peptide strands containing a single isomeric residue in the backbone. Peptide isomerization occurs in long-lived proteins and has been linked with diseases such as Alzheimer's, cataracts and cancer, so isomers are valuable yet underexplored targets for selective recognition. Planar cationic fluorophores can selectively bind hydrophobic, Trp-containing peptide strands in solution, and when paired with receptors that provide a competitive host for the fluorophore, can form a differential sensing array that enables selective discrimination of peptide isomers. Residue variations such as D- and L-Asp, D- and L-isoAsp, D-Ser and D-Glu can all be recognized, simply by their effects on the folded structure of the flexible peptide. Molecular dynamics simulations were applied to determine the most favorable conformation of the peptide : fluorophore conjugate, indicating that favorable π-stacking with internal tryptophan residues in a folded binding pocket enables micromolar binding affinity.

What Strengthens Protein-Protein Interactions: Analysis and Applications of Residue Correlation Networks.

Identifying residues critical to protein-protein binding and efficient design of stable and specific protein binders are challenging tasks. Extending beyond the direct contacts in a protein-protein binding interface, our study employs computational modeling to reveal the essential network of residue interactions and dihedral angle correlations critical in protein-protein recognition. We hypothesized that mutating residues exhibiting highly correlated dynamic motion within the interaction network could efficiently optimize protein-protein interactions to create tight and selective protein binders. We tested this hypothesis using the ubiquitin (Ub) and MERS coronaviral papain-like protease (PLpro) complex, since Ub is a central player in multiple cellular functions and PLpro is an antiviral drug target. Our designed ubiquitin variant (UbV) hosting three mutated residues displayed a ∼3,500-fold increase in functional inhibition relative to wild-type Ub. Further optimization of two C-terminal residues within the Ub network resulted in a KD of 1.5 nM and IC50 of 9.7 nM for the five-point Ub mutant, eliciting 27,500-fold and 5,500-fold enhancements in affinity and potency, respectively, as well as improved selectivity, without destabilizing the UbV structure. Our study highlights residue correlation and interaction networks in protein-protein interactions, and introduces an effective approach to design high-affinity protein binders for cell biology research and future therapeutics.

What Strengthens Protein-Protein Interactions: Analysis and Applications of Residue Correlation Networks.

Identifying critical residues in protein-protein binding and efficiently designing stable and specific protein binders to target another protein is challenging. In addition to direct contacts in a protein-protein binding interface, our study employs computation modeling to reveal the essential network of residue interaction and dihedral angle correlation critical in protein-protein recognition. We propose that mutating residues regions exhibited highly correlated motions within the interaction network can efficiently optimize protein-protein interactions to create tight and selective protein binders. We validated our strategy using ubiquitin (Ub) and MERS coronaviral papain-like protease (PLpro) complexes, where Ub is one central player in many cellular functions and PLpro is an antiviral drug target. Molecular dynamics simulations and experimental assays were used to predict and verify our designed Ub variant (UbV) binders. Our designed UbV with 3 mutated residues resulted in a ~3,500-fold increase in functional inhibition, compared with the wild-type Ub. Further optimization by incorporating 2 more residues within the network, the 5-point mutant achieved a KD of 1.5 nM and IC50 of 9.7 nM. The modification led to a 27,500-fold and 5,500-fold enhancements in affinity and potency, respectively, as well as improved selectivity, without destabilizing the UbV structure. Our study illustrates the importance of residue correlation and interaction networks in protein-protein interaction and introduces a new approach that can effectively design high affinity protein binder for cell biology studies and future therapeutic solution.

What Strengthens Protein-Protein Interactions: Analysis and Applications of Residue Correlation Networks.

Identifying critical residues in protein-protein binding and efficiently designing stable and specific protein binders is challenging. In addition to direct contacts in a protein-protein binding interface, our study employs computation modeling to reveal the essential network of residue interaction and dihedral angle correlation critical in protein-protein recognition. We propose that mutating residues regions exhibited highly correlated motions within the interaction network can efficiently optimize protein-protein interactions to create tight and selective protein binders. We validated our strategy using ubiquitin (Ub) and MERS coronaviral papain-like protease (PLpro) complexes, where Ub is one central player in many cellular functions and PLpro is an antiviral drug target. Our designed UbV with 3 mutated residues resulted in a ~3,500-fold increase in functional inhibition, compared with the wild-type Ub. Further optimization by incorporating 2 more residues within the network, the 5-point mutant achieved a KD of 1.5 nM and IC50 of 9.7 nM. The modification led to a 27,500-fold and 5,500-fold enhancements in affinity and potency, respectively, as well as improved selectivity, without destabilizing the UbV structure. Our study highlights residue correlation and interaction networks in protein-protein interaction, introduces an effective approach to design high affinity protein binders for cell biology and future therapeutics solutions.

Key Amino Acid Residues of Mitochondrial Transcription Factor A Synergize with Abasic (AP) Site Dynamics To Facilitate AP-Lyase Reactions.

Human mitochondrial DNA (mtDNA) encodes 37 essential genes and plays a critical role in mitochondrial and cellular functions. mtDNA is susceptible to damage by endogenous and exogenous chemicals. Damaged mtDNA molecules are counteracted by the redundancy, repair, and degradation of mtDNA. In response to difficult-to-repair or excessive amounts of DNA lesions, mtDNA degradation is a crucial mitochondrial genome maintenance mechanism. Nevertheless, the molecular basis of mtDNA degradation remains incompletely understood. Recently, mitochondrial transcription factor A (TFAM) has emerged as a factor in degrading damaged mtDNA containing abasic (AP) sites. TFAM has AP-lyase activity, which cleaves DNA at AP sites. Human TFAM and its homologs contain a higher abundance of Glu than that of the proteome. To decipher the role of Glu in TFAM-catalyzed AP-DNA cleavage, we constructed TFAM variants and used biochemical assays, kinetic simulations, and molecular dynamics (MD) simulations to probe the functional importance of E187 near a key residue K186. Our previous studies showed that K186 is a primary residue to cleave AP-DNA via Schiff base chemistry. Here, we demonstrate that E187 facilitates ß-elimination, key to AP-DNA strand scission. MD simulations showed that extrahelical confirmation of the AP lesion and the flexibility of E187 in TFAM-DNA complexes facilitate AP-lyase reactions. Together, highly abundant Lys and Glu residues in TFAM promote AP-DNA strand scission, supporting the role of TFAM in AP-DNA turnover and implying the breadth of this process across different species.

Uncovering water effects in protein-ligand recognition: importance in the second hydration shell and binding kinetics.

Developing a ligand with high affinity for a specific protein target is essential for drug design, and water molecules are well known to play a key role in protein-drug recognition. However, predicting the role of particularly ordered water molecules in drug binding remains challenging. Furthermore, hydration free energy contributed from the water network, including the second shell of water molecules, is far from being well studied. In this research we focused on these aspects to accurately and efficiently evaluate water effects in protein-ligand binding affinity. We developed a new strategy using a free-energy calculation method, VM2. We successfully predicted the stable ordered water molecules in a number of protein systems: PDE 10a, HSP90, tryptophan synthase (TRPS), CDK2 and Factor Xa. In some of these, the second shell of water molecules appeared to be critical in protein-ligand binding. We also applied the strategy to largely improve binding free energy calculation using the MM/PBSA method. When applying MM/PBSA alone for two systems, CDK2 and Factor Xa, the computed binding free energy resulted in poor to moderate R2 values with experimental data. However, including water free energy correction greatly improved the free energy calculation. Furthermore, our work helped to explain how xk263 is a 1000 times faster binder to HIVp than ritonavir, a potentially useful tool for investigating binding kinetics. Our studies reveal the importance of fully considering water effects in therapeutic developments in pharmaceutical and biotechnology industries and for fundamental research in protein-ligand recognition.

Multiscale simulation-guided design of enzyme bioconjugates with enhanced catalysis.

Biopolymer-scaffold modification is widely used to enhance enzyme catalysis. A central challenge is predicting the effects of scaffold position on enzyme properties. Here, we use a computational-experimental approach to develop a model for the effects of DNA scaffold position on enzyme kinetics. Using phosphotriesterase modified with a 20bp dsDNA, we demonstrate that conjugation position is as important as the scaffold's chemistry and structure. Multiscale simulations predict the effective substrate concentration increases close to the scaffold, which has µM-strength binding to the substrate. Kinetic analysis shows that the effective concentration that the scaffold provides is best utilized when positioned next to, but not blocking, the active site. At ~5Å distance between scaffold and active site a 7-fold increase in k cat /K M was achieved. A model that accounts for the substrate concentration as well PTE-DNA geometry accurately captures the kinetic enhancements, enabling prediction of the effect across a range of DNA positions.

Binding Kinetics Toolkit for Analyzing Transient Molecular Conformations and Computing Free Energy Landscapes.

Understanding ligand binding kinetics and thermodynamics, which involves investigating the free, transient, and final complex conformations, is important in fundamental studies and applications for chemical and biomedical systems. Examining the important but transient ligand-protein-bound conformations, in addition to experimentally determined structures, also provides a more accurate estimation for drug efficacy and selectivity. Moreover, obtaining the entire picture of the free energy landscape during ligand binding/unbinding processes is critical in understanding binding mechanisms. Here, we present a Binding Kinetics Toolkit (BKiT) that includes several utilities to analyze trajectories and compute a free energy and kinetics profile. BKiT uses principal component space to generate approximated unbinding or conformational transition coordinates for accurately describing and easily visualizing the molecular motions. We implemented a new partitioning approach to assign indexes along the approximated coordinates that can be used as milestones or microstates. The program can generate input files to run many short classical molecular dynamics simulations and uses milestoning theory to construct the free energy profile and estimate binding residence time. We first validated the method with a host-guest system, aspirin unbinding from ß-cyclodextrin, and then applied the protocol to pyrazolourea compounds and cyclin-dependent kinase 8 and cyclin C complexes, a kinase system of pharmacological interest. Overall, our approaches yielded good agreement with published results and suggest ligand design strategies. The computed unbinding free energy landscape also provides a more complete picture of ligand-receptor binding barriers and stable local minima for deepening our understanding of molecular recognition. BKiT is easy to use and has extensible features for future expansion of utilities for postanalysis and calculations.

Allosteric regulation of substrate channeling: Salmonella typhimurium tryptophan synthase.

The regulation of the synthesis of L-tryptophan (L-Trp) in enteric bacteria begins at the level of gene expression where the cellular concentration of L-Trp tightly controls expression of the five enzymes of the Trp operon responsible for the synthesis of L-Trp. Two of these enzymes, trpA and trpB, form an αßßα bienzyme complex, designated as tryptophan synthase (TS). TS carries out the last two enzymatic processes comprising the synthesis of L-Trp. The TS α-subunits catalyze the cleavage of 3-indole D-glyceraldehyde 3'-phosphate to indole and D-glyceraldehyde 3-phosphate; the pyridoxal phosphate-requiring ß-subunits catalyze a nine-step reaction sequence to replace the L-Ser hydroxyl by indole giving L-Trp and a water molecule. Within αß dimeric units of the αßßα bienzyme complex, the common intermediate indole is channeled from the α site to the ß site via an interconnecting 25 Å-long tunnel. The TS system provides an unusual example of allosteric control wherein the structures of the nine different covalent intermediates along the ß-reaction catalytic path and substrate binding to the α-site provide the allosteric triggers for switching the αßßα system between the open (T) and closed (R) allosteric states. This triggering provides a linkage that couples the allosteric conformational coordinate to the covalent chemical reaction coordinates at the α- and ß-sites. This coupling drives the α- and ß-sites between T and R conformations to achieve regulation of substrate binding and/or product release, modulation of the α- and ß-site catalytic activities, prevention of indole escape from the confines of the active sites and the interconnecting tunnel, and synchronization of the α- and ß-site catalytic activities. Here we review recent advances in the understanding of the relationships between structure, function, and allosteric regulation of the complex found in Salmonella typhimurium.

GeomBD3: Brownian Dynamics Simulation Software for Biological and Engineered Systems.

GeomBD3 is a robust Brownian dynamics simulation package designed to easily handle natural or engineered systems in diverse environments and arrangements. The software package described herein allows users to design, execute, and analyze BD simulations. The simulations use all-atom, rigid molecular models that diffuse according to overdamped Langevin dynamics and interact through electrostatic, Lennard-Jones, and ligand desolvation potentials. The program automatically calculates molecular association rates, surface residence times, and association statistics for any number of user-defined association criteria. Users can also extract molecular association pathways, diffusion coefficients, intermolecular interaction energies, intermolecular contact probability maps, and more using the provided supplementary analysis scripts. We detail the use of the package from start to finish and apply it to a protein-ligand system and a large nucleic acid biosensor. GeomBD3 provides a versatile tool for researchers from various disciplines that can aid in rational design of engineered systems or play an explanatory role as a complement to experiments. GeomBD version 3 is available on our website at http://chemcha-gpu0.ucr.edu/geombd3/ and KBbox at https://kbbox.h-its.org/toolbox/methods/molecular-simulation/geombd/.

An Integrated Pharmacological, Structural, and Genetic Analysis of Extracellular Versus Intracellular ROS Production in Neutrophils.

The neutrophil NADPH oxidase produces both intracellular and extracellular reactive oxygen species (ROS). Although oxidase activity is essential for microbial killing, and ROS can act as signaling molecules in the inflammatory process, excessive extracellular ROS directly contributes to inflammatory tissue damage, as well as to cancer progression and immune dysregulation in the tumor microenvironment. How specific signaling pathways contribute to ROS localization is unclear. Here we used a systems pharmacology approach to identify the specific Class I PI3-K isoform p110ß, and PLD1, but not PLD2, as critical regulators of extracellular, but not intracellular ROS production in primary neutrophils. Combined crystallographic and molecular dynamics analysis of the PX domain of the oxidase component p47phox, which binds the lipid products of PI 3-K and PLD, was used to clarify the membrane-binding mechanism and guide the design of mutant mice whose p47phox is unable to bind 3-phosphorylated inositol phospholipids. Neutrophils from these K43A mutant animals were specifically deficient in extracellular, but not intracellular, ROS production, and showed increased dependency on signaling through the remaining PLD1 arm. These findings identify the PX domain of p47phox as a critical integrator of PLD1 and p110ß signaling for extracellular ROS production, and as a potential therapeutic target for modulating tissue damage and extracellular signaling during inflammation.

Ritonavir and xk263 Binding-Unbinding with HIV-1 Protease: Pathways, Energy and Comparison.

Understanding non-covalent biomolecular recognition, which includes drug-protein bound states and their binding/unbinding processes, is of fundamental importance in chemistry, biology, and medicine. Fully revealing the factors that govern the binding/unbinding processes can further assist in designing drugs with desired binding kinetics. HIV protease (HIVp) plays an integral role in the HIV life cycle, so it is a prime target for drug therapy. HIVp has flexible flaps, and the binding pocket can be accessible by a ligand via various pathways. Comparing ligand association and dissociation pathways can help elucidate the ligand-protein interactions such as key residues directly involved in the interaction or specific protein conformations that determine the binding of a ligand under certain pathway(s). Here, we investigated the ligand unbinding process for a slow binder, ritonavir, and a fast binder, xk263, by using unbiased all-atom accelerated molecular dynamics (aMD) simulation with a re-seeding approach and an explicit solvent model. Using ritonavir-HIVp and xk263-HIVp ligand-protein systems as cases, we sampled multiple unbinding pathways for each ligand and observed that the two ligands preferred the same unbinding route. However, ritonavir required a greater HIVp motion to dissociate as compared with xk263, which can leave the binding pocket with little conformational change of HIVp. We also observed that ritonavir unbinding pathways involved residues which are associated with drug resistance and are distal from catalytic site. Analyzing HIVp conformations sampled during both ligand-protein binding and unbinding processes revealed significantly more overlapping HIVp conformations for ritonavir-HIVp rather than xk263-HIVp. However, many HIVp conformations are unique in xk263-HIVp unbinding processes. The findings are consistent with previous findings that xk263 prefers an induced-fit model for binding and unbinding, whereas ritonavir favors a conformation selection model. This study deepens our understanding of the dynamic process of ligand unbinding and provides insights into ligand-protein recognition mechanisms and drug discovery.

Imaging active site chemistry and protonation states: NMR crystallography of the tryptophan synthase α-aminoacrylate intermediate.

NMR-assisted crystallography-the integrated application of solid-state NMR, X-ray crystallography, and first-principles computational chemistry-holds significant promise for mechanistic enzymology: by providing atomic-resolution characterization of stable intermediates in enzyme active sites, including hydrogen atom locations and tautomeric equilibria, NMR crystallography offers insight into both structure and chemical dynamics. Here, this integrated approach is used to characterize the tryptophan synthase α-aminoacrylate intermediate, a defining species for pyridoxal-5'-phosphate-dependent enzymes that catalyze ß-elimination and replacement reactions. For this intermediate, NMR-assisted crystallography is able to identify the protonation states of the ionizable sites on the cofactor, substrate, and catalytic side chains as well as the location and orientation of crystallographic waters within the active site. Most notable is the water molecule immediately adjacent to the substrate ß-carbon, which serves as a hydrogen bond donor to the ε-amino group of the acid-base catalytic residue ßLys87. From this analysis, a detailed three-dimensional picture of structure and reactivity emerges, highlighting the fate of the L-serine hydroxyl leaving group and the reaction pathway back to the preceding transition state. Reaction of the α-aminoacrylate intermediate with benzimidazole, an isostere of the natural substrate indole, shows benzimidazole bound in the active site and poised for, but unable to initiate, the subsequent bond formation step. When modeled into the benzimidazole position, indole is positioned with C3 in contact with the α-aminoacrylate Cß and aligned for nucleophilic attack. Here, the chemically detailed, three-dimensional structure from NMR-assisted crystallography is key to understanding why benzimidazole does not react, while indole does.

Modeling structural interconversion in Alzheimers' amyloid beta peptide with classical and intrinsically disordered protein force fields.

A comprehensive understanding of the aggregation mechanism in amyloid beta 42 (Aß42) peptide is imperative for developing therapeutic drugs to prevent or treat Alzheimer's disease. Because of the high flexibility and lack of native tertiary structures of Aß42, molecular dynamics (MD) simulations may help elucidate the peptide's dynamics with atomic details and collectively improve ensembles not seen in experiments. We applied microsecond-timescale MD simulations to investigate the dynamics and conformational changes of Aß42 by using a newly developed Amber force field (ff14IDPSFF). We compared the ff14IDPSFF and the regular ff14SB force field by examining the conformational changes of two distinct Aß42 monomers in explicit solvent. Conformational ensembles obtained by simulations depend on the force field and initial structure, Aß42α-helix or Aß42ß-strand. The ff14IDPSFF sampled a high ratio of disordered structures and diverse ß-strand secondary structures; in contrast, ff14SB favored helicity during the Aß42α-helix simulations. The conformations obtained from Aß42ß-strand simulations maintained a balanced content in the disordered and helical structures when simulated by ff14SB, but the conformers clearly favored disordered and ß-sheet structures simulated by ff14IDPSFF. The results obtained with ff14IDPSFF qualitatively reproduced the NMR chemical shifts well. In-depth peptide and cluster analysis revealed some characteristic features that may be linked to early onset of the fibril-like structure. The C-terminal region (mainly M35-V40) featured in-registered anti-parallel ß-strand (ß-hairpin) conformations with tested systems. Our work should expand the knowledge of force field and structure dependency in MD simulations and reveals the underlying structural mechanism-function relationship in Aß42 peptides. Communicated by Ramaswamy H. Sarma.

Discovery of antimicrobial agent targeting tryptophan synthase.

Antibiotic resistance is a continually growing challenge in the treatment of various bacterial infections worldwide. New drugs and new drug targets are necessary to curb the threat of infectious diseases caused by multidrug-resistant pathogens. The tryptophan biosynthesis pathway is essential for bacterial growth but is absent in higher animals and humans. Drugs that can inhibit the bacterial biosynthesis of tryptophan offer a new class of antibiotics. In this work, we combined a structure-based strategy using in silico docking screening and molecular dynamics (MD) simulations to identify compounds targeting the α subunit of tryptophan synthase with experimental methods involving the whole-cell minimum inhibitory concentration (MIC) test, solution state NMR, and crystallography to confirm the inhibition of L-tryptophan biosynthesis. Screening 1,800 compounds from the National Cancer Institute Diversity Set I against α subunit revealed 28 compounds for experimental validation; four of the 28 hit compounds showed promising activity in MIC testing. We performed solution state NMR experiments to demonstrate that a one successful inhibitor, 3-amino-3-imino-2-phenyldiazenylpropanamide (Compound 1) binds to the α subunit. We also report a crystal structure of Salmonella enterica serotype Typhimurium tryptophan synthase in complex with Compound 1 which revealed a binding site at the αß interface of the dimeric enzyme. MD simulations were carried out to examine two binding sites for the compound. Our results show that this small molecule inhibitor could be a promising lead for future drug development.

Proteome-Wide Characterizations of N6-Methyl-Adenosine Triphosphate- and N6-Furfuryl-Adenosine Triphosphate-Binding Capabilities of Kinases.

Kinases catalyze the transfer of the γ-phosphate group from adenosine triphosphate (ATP) to their protein and small-molecule substrates, and this phosphorylation is a crucial element of multiple cell signaling pathways. Herein, we employed isotope-coded ATP acyl-phosphate probes, in conjunction with a multiple-reaction monitoring (MRM)-based targeted proteomic method for proteome-wide identifications of endogenous kinases that can bind to two N6-modified ATP derivatives, N6-methyl-ATP (N6-Me-ATP), and N6-furfuryl-ATP (a.k.a. kinetin triphosphate, KTP). We found that, among the ∼300 quantified kinases, 27 and 18 are candidate kinases that can bind to KTP and N6-Me-ATP, respectively. Additionally, GSK3α and GSK3ß are among the kinases that can bind to both ATP analogues. Moreover, the in vitro biochemical assay showed that GSK3ß could employ N6-Me-ATP but not KTP as the phosphate group donor to phosphorylate its substrate peptide. Molecular modeling studies provided insights into the differences between N6-Me-ATP and KTP in enabling the GSK3ß-mediated phosphorylation. Together, our chemoproteomic approach led to the identification of endogenous kinases that can potentially be targeted by the two ATP analogues. The approach should be generally applicable for assessing endogenous kinases targeted by other ATP and purine analogues.

Substitution of a Surface-Exposed Residue Involved in an Allosteric Network Enhances Tryptophan Synthase Function in Cells.

Networks of noncovalent amino acid interactions propagate allosteric signals throughout proteins. Tryptophan synthase (TS) is an allosterically controlled bienzyme in which the indole product of the alpha subunit (αTS) is transferred through a 25 Å hydrophobic tunnel to the active site of the beta subunit (ßTS). Previous nuclear magnetic resonance and molecular dynamics simulations identified allosteric networks in αTS important for its function. We show here that substitution of a distant, surface-exposed network residue in αTS enhances tryptophan production, not by activating αTS function, but through dynamically controlling the opening of the indole channel and stimulating ßTS activity. While stimulation is modest, the substitution also enhances cell growth in a tryptophan-auxotrophic strain of Escherichia coli compared to complementation with wild-type αTS, emphasizing the biological importance of the network. Surface-exposed networks provide new opportunities in allosteric drug design and protein engineering, and hint at potential information conduits through which the functions of a metabolon or even larger proteome might be coordinated and regulated.

Molecular Mechanics Study of Flow and Surface Influence in Ligand-Protein Association.

Ligand-protein association is the first and critical step for many biological and chemical processes. This study investigated the molecular association processes under different environments. In biology, cells have different compartments where ligand-protein binding may occur on a membrane. In experiments involving ligand-protein binding, such as the surface plasmon resonance and continuous flow biosynthesis, a substrate flow and surface are required in experimental settings. As compared with a simple binding condition, which includes only the ligand, protein, and solvent, the association rate and processes may be affected by additional ligand transporting forces and other intermolecular interactions between the ligand and environmental objects. We evaluated these environmental factors by using a ligand xk263 binding to HIV protease (HIVp) with atomistic details. Using Brownian dynamics simulations, we modeled xk263 and HIVp association time and probability when a system has xk263 diffusion flux and a non-polar self-assembled monolayer surface. We also examined different protein orientations and accessible surfaces for xk263. To allow xk263 to access to the dimer interface of immobilized HIVp, we simulated the system by placing the protein 20Å above the surface because immobilizing HIVp on a surface prevented xk263 from contacting with the interface. The non-specific interactions increased the binding probability while the association time remained unchanged. When the xk263 diffusion flux increased, the effective xk263 concentration around HIVp, xk263-HIVp association time and binding probability decreased non-linearly regardless of interacting with the self-assembled monolayer surface or not. The work sheds light on the effects of the solvent flow and surface environment on ligand-protein associations and provides a perspective on experimental design.