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.

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.

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/.

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.

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.

Dynamics and molecular interactions of single-stranded DNA in nucleic acid biosensors with varied surface properties.

Electrochemical DNA biosensors utilizing self-assembled monolayers (SAMs) with inserted DNA probes are promising biosensor designs because of their ease of preparation, miniaturization, and tunability. However, much is still unknown about the interactions between biomolecules such as DNA and various surfaces. A fundamental question regarding these sensors concerns the nature of diffusion of target molecules taking place on sensor surfaces and whether it speeds up the molecular recognition process. Lack of understanding of molecular interaction and surface diffusion in addition to questions regarding the behavior of DNA probes immobilized on these surfaces currently limits the rational design of nucleic acid biosensors. Using all-atom unbiased molecular dynamics (MD) simulations we found that single-stranded DNA (ssDNA) behavior on SAMs is drastically altered by different surface chemistries, with ssDNA adopting very different orientations upon adsorption and surface diffusivity varying over an order of magnitude. Probe behavior varies equally broadly as probes are considerably more stable in certain SAMs than others, which affects the accessibility of probes to the target molecules and likely changes DNA hybridization kinetics in multiple ways. We also found that nearby probes can alter each other's orientations substantially, which highlights the importance of surface density control. Our results elucidate nucleic acid biosensor dynamics vital to rational design and offer insights that can aid in the design of surface properties and patterning for specific applications.

Endosidin2 targets conserved exocyst complex subunit EXO70 to inhibit exocytosis.

The exocyst complex regulates the last steps of exocytosis, which is essential to organisms across kingdoms. In humans, its dysfunction is correlated with several significant diseases, such as diabetes and cancer progression. Investigation of the dynamic regulation of the evolutionarily conserved exocyst-related processes using mutants in genetically tractable organisms such as Arabidopsis thaliana is limited by the lethality or the severity of phenotypes. We discovered that the small molecule Endosidin2 (ES2) binds to the EXO70 (exocyst component of 70 kDa) subunit of the exocyst complex, resulting in inhibition of exocytosis and endosomal recycling in both plant and human cells and enhancement of plant vacuolar trafficking. An EXO70 protein with a C-terminal truncation results in dominant ES2 resistance, uncovering possible distinct regulatory roles for the N terminus of the protein. This study not only provides a valuable tool in studying exocytosis regulation but also offers a potentially new target for drugs aimed at addressing human disease.

Ligand Binding Pathways and Conformational Transitions of the HIV Protease.

It is important to determine the binding pathways and mechanisms of ligand molecules to target proteins to effectively design therapeutic drugs. Molecular dynamics (MD) is a promising computational tool that allows us to simulate protein-drug binding at an atomistic level. However, the gap between the time scales of current simulations and those of many drug binding processes has limited the usage of conventional MD, which has been reflected in studies of the HIV protease. Here, we have applied a robust enhanced simulation method, Gaussian accelerated molecular dynamics (GaMD), to sample binding pathways of the XK263 ligand and associated protein conformational changes in the HIV protease. During two of 10 independent GaMD simulations performed over 500-2500 ns, the ligand was observed to successfully bind to the protein active site. Although GaMD-derived free energy profiles were not fully converged because of insufficient sampling of the complex system, the simulations still allowed us to identify relatively low-energy intermediate conformational states during binding of the ligand to the HIV protease. Relative to the X-ray crystal structure, the XK263 ligand reached a minimum root-mean-square deviation (RMSD) of 2.26 Å during 2.5 µs of GaMD simulation. In comparison, the ligand RMSD reached a minimum of only ∼5.73 Å during an earlier 14 µs conventional MD simulation. This work highlights the enhanced sampling power of the GaMD approach and demonstrates its wide applicability to studies of drug-receptor interactions for the HIV protease and by extension many other target proteins.

Adaptation of a Genetic Screen Reveals an Inhibitor for Mitochondrial Protein Import Component Tim44.

Diverse protein import pathways into mitochondria use translocons on the outer membrane (TOM) and inner membrane (TIM). We adapted a genetic screen, based on Ura3 mistargeting from mitochondria to the cytosol, to identify small molecules that attenuated protein import. Small molecule mitochondrial import blockers of the Carla Koehler laboratory (MB)-10 inhibited import of substrates that require the TIM23 translocon. Mutational analysis coupled with molecular docking and molecular dynamics modeling revealed that MB-10 binds to a specific pocket in the C-terminal domain of Tim44 of the protein-associated motor (PAM) complex. This region was proposed to anchor Tim44 to the membrane, but biochemical studies with MB-10 show that this region is required for binding to the translocating precursor and binding to mtHsp70 in low ATP conditions. This study also supports a direct role for the PAM complex in the import of substrates that are laterally sorted to the inner membrane, as well as the mitochondrial matrix. Thus, MB-10 is the first small molecule modulator to attenuate PAM complex activity, likely through binding to the C-terminal region of Tim44.

Construction of a Sequenceable Protein Mimetic Peptide Library with a True 3D Diversifiable Chemical Space.

We present here a library of protein mimetic bicyclic peptides. These nanosized structures exhibit rigid backbones and spatially diversifiable side chains. They present modular amino acids on all three linkages, providing access to a true 3D diversifiable chemical space. These peptides are synthesized through a Cu-catalyzed click reaction and a Ru-catalyzed ring-closing metathesis reaction. Their bicyclic topology can be reduced to a linear one, using Edman degradation and Pd-catalyzed deallylation reactions. The linearization approaches allow de novo sequencing through mass spectrometry methods. We demonstrate the function of a particular peptide that was identified through a high throughput screening against the E363-R378 epitope on the intrinsically disordered c-Myc oncoprotein. Intracellular delivery of this peptide could interfere with the c-Myc-mediated transcription and inhibit proliferation in a human glioblastoma cell line.

Role of Molecular Interactions and Protein Rearrangement in the Dissociation Kinetics of p38α MAP Kinase Type-I/II/III Inhibitors.

Understanding the governing factors of fast or slow inhibitor binding/unbinding assists in developing drugs with preferred kinetic properties. For inhibitors with the same binding affinity targeting different binding sites of the same protein, the kinetic behavior can profoundly differ. In this study, we investigated unbinding kinetics and mechanisms of fast (type-I) and slow (type-II/III) binders of p38α mitogen-activated protein kinase, where the crystal structures showed that type-I and type-II/III inhibitors bind to pockets with different conformations of the Asp-Phe-Gly (DFG) motif. The work used methods that combine conventional molecular dynamics (MD), accelerated molecular dynamics (AMD) simulations, and the newly developed pathway search guided by internal motions (PSIM) method to find dissociation pathways. The study focuses on revealing key interactions and molecular rearrangements that hinder ligand dissociation by using umbrella sampling and post-MD processing to examine changes in free energy during ligand unbinding. As anticipated, the initial dissociation steps all require breaking interactions that appeared in crystal structures of the bound complexes. Interestingly, for type-I inhibitors such as SB2, p38α keeps barrier-free conformational fluctuation in the ligand-bound complex and during ligand dissociation. In contrast, with a type-II/III inhibitor such as BIRB796, with the rearrangements of p38α in its bound state, ligand unbinding features energetically unfavorable protein-ligand concerted movement. Our results also show that the type-II/III inhibitors preferred dissociation pathways through the allosteric channel, which is consistent with an existing publication. The study suggests that the level of required protein rearrangement is one major determining factor of drug binding kinetics in p38α systems, providing useful information for development of inhibitors.

A molecular dynamics investigation of CDK8/CycC and ligand binding: conformational flexibility and implication in drug discovery.

Abnormal activity of cyclin-dependent kinase 8 (CDK8) along with its partner protein cyclin C (CycC) is a common feature of many diseases including colorectal cancer. Using molecular dynamics (MD) simulations, this study determined the dynamics of the CDK8-CycC system and we obtained detailed breakdowns of binding energy contributions for four type-I and five type-II CDK8 inhibitors. We revealed system motions and conformational changes that will affect ligand binding, confirmed the essentialness of CycC for inclusion in future computational studies, and provide guidance in development of CDK8 binders. We employed unbiased all-atom MD simulations for 500 ns on twelve CDK8-CycC systems, including apoproteins and protein-ligand complexes, then performed principal component analysis (PCA) and measured the RMSF of key regions to identify protein dynamics. Binding pocket volume analysis identified conformational changes that accompany ligand binding. Next, H-bond analysis, residue-wise interaction calculations, and MM/PBSA were performed to characterize protein-ligand interactions and find the binding energy. We discovered that CycC is vital for maintaining a proper conformation of CDK8 to facilitate ligand binding and that the system exhibits motion that should be carefully considered in future computational work. Surprisingly, we found that motion of the activation loop did not affect ligand binding. Type-I and type-II ligand binding is driven by van der Waals interactions, but electrostatic energy and entropic penalties affect type-II binding as well. Binding of both ligand types affects protein flexibility. Based on this we provide suggestions for development of tighter-binding CDK8 inhibitors and offer insight that can aid future computational studies.

Immunoproteasome inhibition and bioactivity of thiasyrbactins.

A family of macrodilactam natural products, the syrbactins, are known proteasome inhibitors. A small group of syrbactin analogs was prepared with a sulfur-for-carbon substitution to enhance synthetic accessibility and facilitate modulation of their solubility. Two of these compounds surprisingly proved to be inhibitors of the trypsin-like catalytic site, including of the immunoproteasome. Their bound and free conformations suggest special properties of the thiasyrbactin ring are responsible for this unusual preference, which may be exploited to develop drug-like immunoproteasome inhibitors. These compounds show greater selectivity than earlier compounds used to infer phenotypes of immunoproteasome inhibition, like ONX-0914.

Mechanism of the Association Pathways for a Pair of Fast and Slow Binding Ligands of HIV-1 Protease.

Equilibrium constants, together with kinetic rate constants of binding, are key factors in the efficacy and safety of drug compounds, informing drug design. However, the association pathways of protein-ligand binding, which contribute to their kinetic behaviors, are little understood. In this work, we used unbiased all-atom molecular dynamics (MD) simulations with an explicit solvent model to study the association processes of protein-ligand binding. Using the HIV protease (HIVp)-xk263 and HIVp-ritonavir protein-ligand systems as cases, we observed that ligand association is a multistep process involving diffusion, localization, and conformational rearrangements of the protein, ligand, and water molecules. Moreover, these two ligands preferred different routes of binding, which reflect two well-known binding mechanisms: induced-fit and conformation selection models. Our study shows that xk263 has a stronger capacity for desolvating surrounding water molecules, thereby inducing a semiopen conformation of the HIVp flaps (induced-fit model). In contrast, the slow dehydration characteristic of ritonavir allows for gradual association with the binding pocket of HIVp when the protein's flap conformation is fully open (conformation selection model). By studying the mechanism of ligand association and understanding the role of solvent molecules during the binding event, we can obtain a different perspective on the mechanism of macromolecule recognition, providing insights into drug discovery.

Syrbactin Structural Analog TIR-199 Blocks Proteasome Activity and Induces Tumor Cell Death.

Multiple myeloma is an aggressive hematopoietic cancer of plasma cells. The recent emergence of three effective FDA-approved proteasome-inhibiting drugs, bortezomib (Velcade®), carfilzomib (Kyprolis®), and ixazomib (Ninlaro®), confirms that proteasome inhibitors are therapeutically useful against neoplastic disease, in particular refractory multiple myeloma and mantle cell lymphoma. This study describes the synthesis, computational affinity assessment, and preclinical evaluation of TIR-199, a natural product-derived syrbactin structural analog. Molecular modeling and simulation suggested that TIR-199 covalently binds each of the three catalytic subunits (ß1, ß2, and ß5) and revealed key interaction sites. In vitro and cell culture-based proteasome activity measurements confirmed that TIR-199 inhibits the proteasome in a dose-dependent manner and induces tumor cell death in multiple myeloma and neuroblastoma cells as well as other cancer types in the NCI-60 cell panel. It is particularly effective against kidney tumor cell lines, with >250-fold higher anti-tumor activities than observed with the natural product syringolin A. In vivo studies in mice revealed a maximum tolerated dose of TIR-199 at 25 mg/kg. The anti-tumor activity of TIR-199 was confirmed in hollow fiber assays in mice. Adverse drug reaction screens in a kidney panel revealed no off-targets of concern. This is the first study to examine the efficacy of a syrbactin in animals. Taken together, the results suggest that TIR-199 is a potent new proteasome inhibitor with promise for further development into a clinical drug for the treatment of multiple myeloma and other forms of cancer.

Visualizing the tunnel in tryptophan synthase with crystallography: Insights into a selective filter for accommodating indole and rejecting water.

Four new X-ray structures of tryptophan synthase (TS) crystallized with varying numbers of the amphipathic N-(4'-trifluoromethoxybenzoyl)-2-aminoethyl phosphate (F6) molecule are presented. These structures show one of the F6 ligands threaded into the tunnel from the ß-site and reveal a distinct hydrophobic region. Over this expanse, the interactions between F6 and the tunnel are primarily nonpolar, while the F6 phosphoryl group fits into a polar pocket of the ß-subunit active site. Further examination of TS structures reveals that one portion of the tunnel (T1) binds clusters of water molecules, whereas waters are not observed in the nonpolar F6 binding region of the tunnel (T2). MD simulation of another TS structure with an unobstructed tunnel also indicates the T2 region of the tunnel excludes water, consistent with a dewetted state that presents a significant barrier to the transfer of water into the closed ß-site. We conclude that hydrophobic molecules can freely diffuse between the α- and ß-sites via the tunnel, while water does not. We propose that exclusion of water serves to inhibit reaction of water with the α-aminoacrylate intermediate to form ammonium ion and pyruvate, a deleterious side reaction in the αß-catalytic cycle. Finally, while most TS structures show ßPhe280 partially blocking the tunnel between the α- and ß-sites, new structures show an open tunnel, suggesting the flexibility of the ßPhe280 side chain. Flexible docking studies and MD simulations confirm that the dynamic behavior of ßPhe280 allows unhindered transfer of indole through the tunnel, therefore excluding a gating role for this residue.

Characterization of Promiscuous Binding of Phosphor Ligands to Breast-Cancer-Gene 1 (BRCA1) C-Terminal (BRCT): Molecular Dynamics, Free Energy, Entropy and Inhibitor Design.

Inhibition of the protein-protein interaction (PPI) mediated by breast-cancer-gene 1 C-terminal (BRCT) is an attractive strategy to sensitize breast and ovarian cancers to chemotherapeutic agents that induce DNA damage. Such inhibitors could also be used for studies to understand the role of this PPI in DNA damage response. However, design of BRCT inhibitors is challenging because of the inherent flexibility associated with this domain. Several studies identified short phosphopeptides as tight BRCT binders. Here we investigated the thermodynamic properties of 18 phosphopeptides or peptide with phosphate mimic and three compounds with phosphate groups binding to BRCT to understand promiscuous molecular recognition and guide inhibitor design. We performed molecular dynamics (MD) simulations to investigate the interactions between inhibitors and BRCT and their dynamic behavior in the free and bound states. MD simulations revealed the key role of loops in altering the shape and size of the binding site to fit various ligands. The mining minima (M2) method was used for calculating binding free energy to explore the driving forces and the fine balance between configuration entropy loss and enthalpy gain. We designed a rigidified ligand, which showed unfavorable experimental binding affinity due to weakened enthalpy. This was because it lacked the ability to rearrange itself upon binding. Investigation of another phosphate group containing compound, C1, suggested that the entropy loss can be reduced by preventing significant narrowing of the energy well and introducing multiple new compound conformations in the bound states. From our computations, we designed an analog of C1 that introduced new intermolecular interactions to strengthen attractions while maintaining small entropic penalty. This study shows that flexible compounds do not always encounter larger entropy penalty, compared with other more rigid binders, and highlights a new strategy for inhibitor design.

Mechanisms of Enhanced Catalysis in Enzyme-DNA Nanostructures Revealed through Molecular Simulations and Experimental Analysis.

Understanding and controlling the molecular interactions between enzyme substrates and DNA nanostructures has important implications in the advancement of enzyme-DNA technologies as solutions in biocatalysis. Such hybrid nanostructures can be used to create enzyme systems with enhanced catalysis by controlling the local chemical and physical environments and the spatial organization of enzymes. Here we have used molecular simulations with corresponding experiments to describe a mechanism of enhanced catalysis due to locally increased substrate concentrations. With a series of DNA nanostructures conjugated to horseradish peroxidase, we show that binding interactions between substrates and the DNA structures can increase local substrate concentrations. Increased local substrate concentrations in HRP(DNA) nanostructures resulted in 2.9- and 2.4-fold decreases in the apparent Michaelis constants of tetramethylbenzidine and 4-aminophenol, substrates of HRP with tunable binding interactions to DNA nanostructures with dissociation constants in the micromolar range. Molecular simulations and kinetic analysis also revealed that increased local substrate concentrations enhanced the rates of substrate association. Identification of the mechanism of increased local concentration of substrates in close proximity to enzymes and their active sites adds to our understanding of nanostructured biocatalysis from which we can develop guidelines for enhancing catalysis in rationally designed systems.

Amino acid networks in a (ß/α)8 barrel enzyme change during catalytic turnover.

Proteins can be viewed as small-world networks of amino acid residues connected through noncovalent interactions. Nuclear magnetic resonance chemical shift covariance analyses were used to identify long-range amino acid networks in the α subunit of tryptophan synthase both for the resting state (in the absence of substrate and product) and for the working state (during catalytic turnover). The amino acid networks observed stretch from the surface of the protein into the active site and are different between the resting and working states. Modification of surface residues on the network alters the structural dynamics of active-site residues over 25 Å away and leads to changes in catalytic rates. These findings demonstrate that amino acid networks, similar to those studied here, are likely important for coordinating structural changes necessary for enzyme function and regulation.

Protonation states of the tryptophan synthase internal aldimine active site from solid-state NMR spectroscopy: direct observation of the protonated Schiff base linkage to pyridoxal-5'-phosphate.

The acid-base chemistry that drives catalysis in pyridoxal-5'-phosphate (PLP)-dependent enzymes has been the subject of intense interest and investigation since the initial identification of PLP's role as a coenzyme in this extensive class of enzymes. It was first proposed over 50 years ago that the initial step in the catalytic cycle is facilitated by a protonated Schiff base form of the holoenzyme in which the linking lysine ε-imine nitrogen, which covalently binds the coenzyme, is protonated. Here we provide the first (15)N NMR chemical shift measurements of such a Schiff base linkage in the resting holoenzyme form, the internal aldimine state of tryptophan synthase. Double-resonance experiments confirm the assignment of the Schiff base nitrogen, and additional (13)C, (15)N, and (31)P chemical shift measurements of sites on the PLP coenzyme allow a detailed model of coenzyme protonation states to be established.