From Steroid and Drug Metabolism to Glycobiology, Using Sulfotransferase Structures to Understand and Tailor Function.

Sulfotransferases are ubiquitous enzymes that transfer a sulfo group from the universal cofactor donor 3'-phosphoadenosine 5'-phosphosulfate to a broad range of acceptor substrates. In humans, the cytosolic sulfotransferases are involved in the sulfation of endogenous compounds such as steroids, neurotransmitters, hormones, and bile acids as well as xenobiotics including drugs, toxins, and environmental chemicals. The Golgi associated membrane-bound sulfotransferases are involved in post-translational modification of macromolecules from glycosaminoglycans to proteins. The sulfation of small molecules can have profound biologic effects on the functionality of the acceptor, including activation, deactivation, or enhanced metabolism and elimination. Sulfation of macromolecules has been shown to regulate a number of physiologic and pathophysiological pathways by enhancing binding affinity to regulatory proteins or binding partners. Over the last 25 years, crystal structures of these enzymes have provided a wealth of information on the mechanisms of this process and the specificity of these enzymes. This review will focus on the general commonalities of the sulfotransferases, from enzyme structure to catalytic mechanism as well as providing examples into how structural information is being used to either design drugs that inhibit sulfotransferases or to modify the enzymes to improve drug synthesis. SIGNIFICANCE STATEMENT: This manuscript honors Dr. Masahiko Negishi's contribution to the understanding of sulfotransferase mechanism, specificity, and roles in biology by analyzing the crystal structures that have been solved over the last 25 years.

Preferential DNA Polymerase ß Reverse Reaction with Imidodiphosphate.

DNA replication and repair reactions involve the addition of a deoxynucleoside monophosphate onto a growing DNA strand with the loss of pyrophosphate. This chemical reaction is also reversible; the addition of pyrophosphate generates a deoxynucleoside triphosphate, thereby shortening the DNA by one nucleotide. The forward DNA synthesis and reverse pyrophosphorolysis reactions strictly require the presence of divalent metals, usually magnesium, at the reactive center as cofactors. The overall equilibrium enzymatic reaction strongly favors DNA synthesis over pyrophosphorolysis with natural substrates. The DNA polymerase ß chemical reaction has been structurally and kinetically characterized, employing natural and chemically modified substrates. Substituting an imido-moiety (NH) for the bridging oxygen between Pß and Pγ of dGTP dramatically decreased the overall enzymatic activity and resulted in a chemical equilibrium that strongly favors the reverse reaction (i.e., K ≪ 1). Using QM/MM calculations in conjunction with the utilization of parameters such as quantum mechanically derived atomic charges, we have examined the chemical foundation for the altered equilibrium with this central biological reaction. The calculations indicate that the rapid reverse reaction is likely due, in part, to the increased nucleophilicity of the reactive oxygen on the tautomeric form of imidodiphosphate.

Dysfunctional endogenous FIX impairs prophylaxis in a mouse hemophilia B model.

Factor IX (FIX) binds to collagen IV (Col4) in the subendothelial basement membrane. In hemophilia B, this FIX-Col4 interaction reduces the plasma recovery of infused FIX and plays a role in hemostasis. Studies examining the recovery of infused BeneFix (FIXWT) in null (cross-reactive material negative, CRM-) hemophilia B mice suggest the concentration of Col4 readily available for binding FIX is ∼405 nM with a 95% confidence interval of 374 to 436 nM. Thus, the vascular cache of FIX bound to Col4 is several-fold the FIX level measured in plasma. In a mouse model of prophylactic therapy (testing hemostasis by saphenous vein bleeding 7 days after infusion of 150 IU/kg FIX), FIXWT and the increased half-life FIXs Alprolix (FIXFC) and Idelvion (FIXAlb) produce comparable hemostatic results in CRM- mice. In bleeding CRM- hemophilia B mice, the times to first clot at a saphenous vein injury site after the infusions of the FIX agents are significantly different, at FIXWT < FIXFC < FIXAlb Dysfunctional forms of FIX, however, circulate in the majority of patients with hemophilia B (CRM+). In the mouse prophylactic therapy model, none of the FIX products improves hemostasis in CRM+ mice expressing a dysfunctional FIX, FIXR333Q, that nevertheless competes with infused FIX for Col4 binding and potentially other processes involving FIX. The results in this mouse model of CRM+ hemophilia B demonstrate that the endogenous expression of a dysfunctional FIX can deleteriously affect the hemostatic response to prophylactic therapy.

Warfarin and vitamin K epoxide reductase: a molecular accounting for observed inhibition.

Vitamin K epoxide reductase (VKOR), an endoplasmic reticulum membrane protein, is the key enzyme for vitamin K-dependent carboxylation, a posttranslational modification that is essential for the biological functions of coagulation factors. VKOR is the target of the most widely prescribed oral anticoagulant, warfarin. However, the topological structure of VKOR and the mechanism of warfarin's inhibition of VKOR remain elusive. Additionally, it is not clear why warfarin-resistant VKOR mutations identified in patients significantly decrease warfarin's binding affinity, but have only a minor effect on vitamin K binding. Here, we used immunofluorescence confocal imaging of VKOR in live mammalian cells and PEGylation of VKOR's endogenous cytoplasmic-accessible cysteines in intact microsomes to probe the membrane topology of human VKOR. Our results show that the disputed loop sequence between the first and second transmembrane (TM) domain of VKOR is located in the cytoplasm, supporting a 3-TM topological structure of human VKOR. Using molecular dynamics (MD) simulations, a T-shaped stacking interaction between warfarin and tyrosine residue 139, within the proposed TY139A warfarin-binding motif, was observed. Furthermore, a reversible dynamic warfarin-binding pocket opening and conformational changes were observed when warfarin binds to VKOR. Several residues (Y25, A26, and Y139) were found essential for warfarin binding to VKOR by MD simulations, and these were confirmed by the functional study of VKOR and its mutants in their native milieu using a cell-based assay. Our findings provide new insights into the dynamics of the binding of warfarin to VKOR, as well as into warfarin's mechanism of anticoagulation.

Revealing the role of the product metal in DNA polymerase ß catalysis.

DNA polymerases catalyze a metal-dependent nucleotidyl transferase reaction during extension of a DNA strand using the complementary strand as a template. The reaction has long been considered to require two magnesium ions. Recently, a third active site magnesium ion was identified in some DNA polymerase product crystallographic structures, but its role is not known. Using quantum mechanical/ molecular mechanical calculations of polymerase ß, we find that a third magnesium ion positioned near the newly identified product metal site does not alter the activation barrier for the chemical reaction indicating that it does not have a role in the forward reaction. This is consistent with time-lapse crystallographic structures following insertion of Sp-dCTPαS. Although sulfur substitution deters product metal binding, this has only a minimal effect on the rate of the forward reaction. Surprisingly, monovalent sodium or ammonium ions, positioned in the product metal site, lowered the activation barrier. These calculations highlight the impact that an active site water network can have on the energetics of the forward reaction and how metals or enzyme side chains may interact with the network to modulate the reaction barrier. These results also are discussed in the context of earlier findings indicating that magnesium at the product metal position blocks the reverse pyrophosphorolysis reaction.

Hiding in Plain Sight: The Bimetallic Magnesium Covalent Bond in Enzyme Active Sites.

The transfer of phosphate groups is an essential function of many intracellular biological enzymes. The transfer is in many cases facilitated by a protein scaffold involving two closely spaced magnesium "ions". It has long been a mystery how these "ions" can retain their closely spaced positions throughout enzymatic phosphate transfer: Coulomb's law would dictate large repulsive forces between these ions at the observed distances. Here we show, however, that the electron density can be borrowed from nearby electron-rich oxygens to populate a bonding molecular orbital that is largely localized between the magnesium "ions". The result is that the Mg-Mg core of these phosphate transfer enzymes is surprisingly similar to a metastable [Mg2]2+ ion in the gas phase, an ion that has been identified experimentally and studied with high-level quantum-mechanical calculations. This similarity is confirmed by comparative computations of the electron densities of [Mg2]2+ in the gas phase and the Mg-Mg core in the structures derived from QM/MM studies of high-resolution X-ray crystal structures. That there is a level of covalent bonding between the two Mg "ions" at the core of these enzymes is a novel concept that enables an improved vision of how these enzymes function at the molecular level. The concept is broader than magnesium-other biologically relevant metals (e.g., Mn and Zn) can also form similar stabilizing covalent Me-Me bonds in both organometallic and inorganic crystals.

Requirement for transient metal ions revealed through computational analysis for DNA polymerase going in reverse.

DNA polymerases facilitate faithful insertion of nucleotides, a central reaction occurring during DNA replication and repair. DNA synthesis (forward reaction) is "balanced," as dictated by the chemical equilibrium by the reverse reaction of pyrophosphorolysis. Two closely spaced divalent metal ions (catalytic and nucleotide-binding metals) provide the scaffold for these reactions. The catalytic metal lowers the pKa of O3' of the growing primer terminus, and the nucleotide-binding metal facilitates substrate binding. Recent time-lapse crystallographic studies of DNA polymerases have identified an additional metal ion (product metal) associated with pyrophosphate formation, leading to the suggestion of its possible involvement in the reverse reaction. Here, we establish a rationale for a role of the product metal using quantum mechanical/molecular mechanical calculations of the reverse reaction in the confines of the DNA polymerase ß active site. Additionally, site-directed mutagenesis identifies essential residues and metal-binding sites necessary for pyrophosphorolysis. The results indicate that the catalytic metal site must be occupied by a magnesium ion for pyrophosphorolysis to occur. Critically, the product metal site is occupied by a magnesium ion early in the pyrophosphorolysis reaction path but must be removed later. The proposed dynamic nature of the active site metal ions is consistent with crystallographic structures. The transition barrier for pyrophosphorolysis was estimated to be significantly higher than that for the forward reaction, consistent with kinetic activity measurements of the respective reactions. These observations provide a framework to understand how ions and active site changes could modulate the internal chemical equilibrium of a reaction that is central to genome stability.

Do the crystallographic forms of prethrombin-2 revert to a single form in solution?

It has been earlier established (Pozzi et al. Biochemistry 50 (2011) 10195-10202) that prethrombin-2 crystallizes into two similar but distinct forms: a collapsed form and an alternative form. We employed long molecular dynamics (MD) simulations for these two forms to obtain solvent-equilibrated forms. We find that, at 200ns, the simulated solution collapsed form is quite similar to the X-ray crystal collapsed form, while the simulated solution alternative form deviates from the X-ray crystal alternative form as well as from the solution collapsed form. A detailed structural analysis suggests that the fluctuation of the 140s-loop, in cross-talk with the 220s-loop, may alter the conformation of the W215-E217 segment near the nascent thrombin active site. A rationale is provided for the manner in which interactions of prethrombin-2 with FVa may affect the equilibrium between the two forms of prethrombin-2.

A model for the unique role of factor Va A2 domain extension in the human ternary thrombin-generating complex.

An all-atom human ternary model for the prothrombinase-prothrombin complex, including metal ions and post-translationally modified residues, was constructed from existing X-ray crystal structures. The factor Xa-prothrombin interface was taken from an existing ternary model, which locates the active site of factor Xa in the vicinity of prothrombin cleavage positions. The three sulfotyrosine residues at the C-terminal sequence of factor Va A2 domain are accommodated by modelling rational interactions with positively charged patches on the surface of prothrombin. The entire model is then solvent-equilibrated with molecular dynamics. This ternary model for the thrombin-generating complex provides an estimate as to the role of the C-terminus of the factor Va A2 domain: to establish an interface between FXa and prothrombin and to stabilize the orientation of this interface.

Applications of quantum mechanical/molecular mechanical methods to the chemical insertion step of DNA and RNA polymerization.

We review theoretical attempts to model the chemical insertion reactions of nucleoside triphosphates catalyzed by the nucleic acid polymerases using combined quantum mechanical/molecular mechanical methodology. Due to an existing excellent database of high-resolution X-ray crystal structures, the DNA polymerase ß system serves as a useful template for discussion and comparison. The convergence of structures of high-quality complexes and continued developments of theoretical techniques suggest a bright future for understanding the global features of nucleic acid polymerization.

Analysis on long-range residue-residue communication using molecular dynamics.

We investigated the possibility of inter-residue communication of side chains in barstar, an 89 residue protein, using mutual information theory. The normalized mutual information (NMI) of the dihedral angles of the side chains was obtained from all-atom molecular dynamics simulations. The accumulated NMI from an explicit solvent equilibrated trajectory (600 ns) with free backbone exhibits a parabola-shaped distribution over the inter-residue distances (0-36 Å): smaller at the end regimes but larger in the middle regime. This analysis, plus several other measures, does not find unusual long-range communication for free backbone in explicit solvent simulations.

Amino acid substitution in the active site of DNA polymerase ß explains the energy barrier of the nucleotidyl transfer reaction.

DNA polymerase ß (pol ß) is a bifunctional enzyme widely studied for its roles in base excision DNA repair, where one key function is gap-filling DNA synthesis. In spite of significant progress in recent years, the atomic level mechanism of the DNA synthesis reaction has remained poorly understood. Based on crystal structures of pol ß in complex with its substrates and theoretical considerations of amino acids and metals in the active site, we have proposed that a nearby carboxylate group of Asp256 enables the reaction by accepting a proton from the primer O3'group, thus activating O3'as the nucleophile in the reaction path. Here, we tested this proposal by altering the side chain of Asp256 to Glu and then exploring the impact of this conservative change on the reaction. The D256E enzyme is more than 1000-fold less active than the wild-type enzyme, and the crystal structures are subtly different in the active sites of the D256E and wild-type enzymes. Theoretical analysis of DNA synthesis by the D256E enzyme shows that the O3'proton still transfers to the nearby carboxylate of residue 256. However, the electrostatic stabilization and location of the O3' proton transfer during the reaction path are dramatically altered compared with wild-type. Surprisingly, this is due to repositioning of the Arg254 side chain in the Glu256 enzyme active site, such that Arg254 is not in position to stabilize the proton transfer from O3'. The theoretical results with the wild-type enzyme indicate an early charge reorganization associated with the O3' proton transfer, and this does not occur in the D256E enzyme. The charge reorganization is mediated by the catalytic magnesium ion in the active site.

Weakly Antiferromagentic Coupling Via Superexchange Interaction Between Mn(II)-Mn(II) Atoms: A QM/MM Study of the Active Site of Human Cytosolic X-Propyl Aminopeptidase P.

We investigate the dinuclear manganese, Mn(II)-Mn(II), active site of human cytosolic X-propyl aminopeptidase (XPNPEP1) employing the QM/MM method. The optimized structure supports two manganese atoms at the active site and excludes the possibility of a single Mn(II) atom or other combination of divalent metal ions: Ca(II), Fe(II), Mg(II). A broken symmetry solution verifies an antiferromagnetically coupled state between the Mn(II)-Mn(II) pair, which is the ground state. From the energy difference between the high spin state (HS) and the broken symmetry state (BS), we estimate the exchange coupling constant, J, to be 5.15 cm(-1). Also, we observe multiple bridges (p orbitals) from solvent and two carboxylate linking to the Mn(II)-Mn(II), which leads to the weakly antiferromagnetic interaction of d(5)-d(5) electrons through superexchange coupling.

HPAM: Hirshfeld Partitioned Atomic Multipoles.

An implementation of the Hirshfeld (HD) and Hirshfeld-Iterated (HD-I) atomic charge density partitioning schemes is described. Atomic charges and atomic multipoles are calculated from the HD and HD-I atomic charge densities for arbitrary atomic multipole rank l(max) on molecules of arbitrary shape and size. The HD and HD-I atomic charges/multipoles are tested by comparing molecular multipole moments and the electrostatic potential (ESP) surrounding a molecule with their reference ab initio values. In general, the HD-I atomic charges/multipoles are found to better reproduce ab initio electrostatic properties over HD atomic charges/multipoles. A systematic increase in precision for reproducing ab initio electrostatic properties is demonstrated by increasing the atomic multipole rank from l(max) = 0 (atomic charges) to l(max) = 4 (atomic hexadecapoles). Both HD and HD-I atomic multipoles up to rank l(max) are shown to exactly reproduce ab initio molecular multipole moments of rank L for L ≤ l(max). In addition, molecular dipole moments calculated by HD, HD-I, and ChelpG atomic charges only (l(max) = 0) are compared with reference ab initio values. Significant errors in reproducing ab initio molecular dipole moments are found if only HD or HD-I atomic charges used.

A finite field method for calculating molecular polarizability tensors for arbitrary multipole rank.

A finite field method for calculating spherical tensor molecular polarizability tensors α(lm;l'm') = ∂Δ(lm)/∂ϕ(l'm')* by numerical derivatives of induced molecular multipole Δ(lm) with respect to gradients of electrostatic potential ϕ(l'm')* is described for arbitrary multipole ranks l and l'. Interconversion formulae for transforming multipole moments and polarizability tensors between spherical and traceless Cartesian tensor conventions are derived. As an example, molecular polarizability tensors up to the hexadecapole-hexadecapole level are calculated for water using the following ab initio methods: Hartree-Fock (HF), Becke three-parameter Lee-Yang-Parr exchange-correlation functional (B3LYP), Møller-Plesset perturbation theory up to second order (MP2), and Coupled Cluster theory with single and double excitations (CCSD). In addition, intermolecular electrostatic and polarization energies calculated by molecular multipoles and polarizability tensors are compared with ab initio reference values calculated by the Reduced Variation Space method for several randomly oriented small molecule dimers separated by a large distance. It is discussed how higher order molecular polarizability tensors can be used as a tool for testing and developing new polarization models for future force fields.

Molecular insights into DNA polymerase deterrents for ribonucleotide insertion.

DNA polymerases can misinsert ribonucleotides that lead to genomic instability. DNA polymerase ß discourages ribonucleotide insertion with the backbone carbonyl of Tyr-271; alanine substitution of Tyr-271, but not Phe-272, resulted in a >10-fold loss in discrimination. The Y271A mutant also inserted ribonucleotides more efficiently than wild type on a variety of ribonucleoside (rNMP)-containing DNA substrates. Substituting Mn(2+) for Mg(2+) decreased sugar discrimination for both wild-type and mutant enzymes primarily by increasing the affinity for rCTP. This facilitated crystallization of ternary substrate complexes of both the wild-type and Y271A mutant enzymes. Crystallographic structures of Y271A- and wild type-substrate complexes indicated that rCTP is well accommodated in the active site but that O2' of rCTP and the carbonyl oxygen of Tyr-271 or Ala-271 are unusually close (∼2.5 and 2.6 Å, respectively). Structure-based modeling indicates that the local energetic cost of positioning these closely spaced oxygens is ∼2.2 kcal/mol for the wild-type enzyme. Because the side chain of Tyr-271 also hydrogen bonds with the primer terminus, loss of this interaction affects its catalytic positioning. Our results support a model where DNA polymerase ß utilizes two strategies, steric and geometric, with a single protein residue to deter ribonucleotide insertion.

Lee Pedersen's work in theoretical and computational chemistry and biochemistry.

Nature at the lab level in biology and chemistry can be described by the application of quantum mechanics. In many cases, a reasonable approximation to quantum mechanics is classical mechanics realized through Newton's equations of motion. Dr. Pedersen began his career using quantum mechanics to describe the properties of small molecular complexes that could serve as models for biochemical systems. To describe large molecular systems required a drop-back to classical means and this led surprisingly to a major improvement in the classical treatment of electrostatics for all molecules, not just biological molecules. Recent work has involved the application of quantum mechanics for the putative active sites of enzymes to gain greater insight into the key steps in enzyme catalysis.

A hetero-dimer model for concerted action of vitamin K carboxylase and vitamin K reductase in vitamin K cycle.

Vitamin K carboxylase (VKC) is believed to convert vitamin K, in the vitamin K cycle, to an alkoxide-epoxide form which then reacts with CO(2) and glutamate to generate γ-carboxyglutamic acid (Gla). Subsequently, vitamin K epoxide reductase (VKOR) is thought to convert the alkoxide-epoxide to a hydroquinone form. By recycling vitamin K, the two integral-membrane proteins, VKC and VKOR, maintain vitamin K levels and sustain the blood coagulation cascade. Unfortunately, NMR or X-ray crystal structures of the two proteins have not been characterized. Thus, our understanding of the vitamin K cycle is only partial at the molecular level. In this study, based on prior biochemical experiments on VKC and VKOR, we propose a hetero-dimeric form of VKC and VKOR that may explain the efficient oxidation and reduction of vitamin K during the vitamin K cycle.

Modeling of the DNA-binding site of yeast Pms1 by mass spectrometry.

Mismatch repair (MMR) corrects replication errors that would otherwise lead to mutations and, potentially, various forms of cancer. Among several proteins required for eukaryotic MMR, MutLα is a heterodimer comprised of Mlh1 and Pms1. The two proteins dimerize along their C-terminal domains (CTDs), and the CTD of Pms1 houses a latent endonuclease that is required for MMR. The highly conserved N-terminal domains (NTDs) independently bind DNA and possess ATPase active sites. Here we use two protein footprinting techniques, limited proteolysis and oxidative surface mapping, coupled with mass spectrometry to identify amino acids involved along the DNA-binding surface of the Pms1-NTD. Limited proteolysis experiments elucidated several basic residues that were protected in the presence of DNA, while oxidative surface mapping revealed one residue that is uniquely protected from oxidation. Furthermore, additional amino acids distributed throughout the Pms1-NTD were protected from oxidation either in the presence of a non-hydrolyzable analog of ATP or DNA, indicating that each ligand stabilizes the protein in a similar conformation. Based on the recently published X-ray crystal structure of yeast Pms1-NTD, a model of the Pms1-NTD/DNA complex was generated using the mass spectrometric data as constraints. The proposed model defines the DNA-binding interface along a positively charged groove of the Pms1-NTD and complements prior mutagenesis studies of Escherichia coli and eukaryotic MutL.