Brian J. Bahnson

Crystal Structure of Human Group X Secreted Phospholipase A2 ELECTROSTATICALLY NEUTRAL INTERFACIAL BINDING SURFACE TARGETS ZWITTERIONIC MEMBRANES

The crystal structure of human group X (hGX) secreted phospholipase A2 (sPLA2) has been solved to a resolution of 1.97 Å. As expected the protein fold is similar to previously reported sPLA2 structures. The active site architecture, including the positions of the catalytic residues and the first and second shell water around the Ca2+ cofactor, are highly conserved and remarkably similar to the group IB and group IIA enzymes. Differences are seen in the structures following the (1–12)-N-terminal helix and at the C terminus. These regions are proposed to interact with the substrate membrane surface. The opening to the active site slot is considerably larger in hGX than in human group IIA sPLA2. Furthermore, the electrostatic surface potential of the hGX interfacial-binding surface does not resemble that of the human group IIA sPLA2; the former is highly neutral, whereas the latter is highly cationic. The cationic residues on this face of group IB and IIA enzymes have been implicated in membrane binding and ink cat * allostery. In contrast, hGX does not show activation by the anionic charge at the lipid interface when acting on phospholipid vesicles or short-chain phospholipid micelles. Together, the crystal structure and kinetic results of hGX supports the conclusion that it is as active on zwitterionic as on anionic interfaces, and thus it is predicted to target the zwitterionic membrane surfaces of mammalian cells.

Crystal Structure of Human Plasma Platelet-activating Factor Acetylhydrolase STRUCTURAL IMPLICATION TO LIPOPROTEIN BINDING AND CATALYSIS

Human plasma platelet-activating factor (PAF) acetylhydrolase functions by reducing PAF levels as a general anti-inflammatory scavenger and is linked to anaphylactic shock, asthma, and allergic reactions. The enzyme has also been implicated in hydrolytic activities of other pro-inflammatory agents, such as sn-2 oxidatively fragmented phospholipids. This plasma enzyme is tightly bound to low and high density lipoprotein particles and is also referred to as lipoprotein-associated phospholipase A2. The crystal structure of this enzyme has been solved from x-ray diffraction data collected to a resolution of 1.5Å. It has a classic lipase α/β-hydrolase fold, and it contains a catalytic triad of Ser273, His351, and Asp296. Two clusters of hydrophobic residues define the probable interface-binding region, and a prediction is given of how the enzyme is bound to lipoproteins. Additionally, an acidic patch of 10 carboxylate residues and a neighboring basic patch of three residues are suggested to play a role in high density lipoprotein/low density lipoprotein partitioning. A crystal structure is also presented of PAF acetylhydrolase reacted with the organophosphate compound paraoxon via its active site Ser273. The resulting diethyl phosphoryl complex was used to model the tetrahedral intermediate of the substrate PAF to the active site. The model of interface binding begins to explain the known specificity of lipoprotein-bound substrates and how the active site can be both close to the hydrophobic-hydrophilic interface and at the same time be accessible to the aqueous phase.

[14] Hydrogen tunneling in enzyme catalysis

Publisher Summary An essential part of the understanding of enzyme catalysis is an experimental description of the mechanism, transition state structure, and potential energy surface of the reaction. A semiclassical picture of the reaction can be described from primary and secondary deuterium kinetic isotope effects. However, the isotopes of hydrogen [protium (H), deuterium (D), and tritium (T)] have de Broglie wavelengths that are similar to the distances they must typically travel during a hydrogen transfer reaction. This property has led to the recognition that the behavior of hydrogen is poised between classical and quantum mechanical realms. To describe the enzyme-catalyzed hydrogen transfers rigorously, these reactions need to be examined for quantum effects. Hydrogen tunneling has been shown to be a general feature of a variety of enzyme systems that have been examined. Some features of a chemical reaction that affect the tunneling probability and have been observed to be altered in enzyme-catalyzed reactions are the degree of participation of solvent reorganization, the thermodynamic relationship between substrates and products, and the height and width of the reaction coordinate energy barrier. The optimization of enzyme catalysis may entail the evolutionary implementation of these chemical strategies that increase the probability of tunneling, and thereby accelerate the reaction rate. Additionally, the exploration of variations in the extent of hydrogen tunneling offers a powerful new approach in determining how protein structure is linked to the optimization of enzyme catalysis.

Predictive crystallization of ribonuclease A via rapid screening of osmotic second virial coefficients

Important progress has been made in recent years toward developing a molecular‐level understanding of protein phase behavior in terms of the osmotic second virial coefficient, a thermodynamic parameter that characterizes pairwise protein interactions. Yet there has been little practical application of this knowledge to the field of protein crystallization, largely because of the difficult and time‐consuming nature of traditional techniques for characterizing protein interactions. Self‐interaction chromatography has recently been proposed as a highly efficient method for measuring the osmotic second virial coefficient. The utility of the technique is examined in this work by characterizing virial coefficients for ribonuclease A under 59 solution conditions using several crystallization additives, including PEG, sodium chloride, ammonium sulfate, and propanol. The virial coefficient measurements show some counterintuitive trends and shed light on the previous difficulties in crystallizing ribonuclease A. Crystallization experiments at the corresponding solution conditions were conducted by using ultracentrifugal crystallization. Using this methodology, ribonuclease A crystals were obtained under conditions for which the virial coefficients fell within the “crystallization slot.” Crystallographic characterization showed that the crystals diffract to high resolution. Metastable crystals were also obtained for conditions outside, but near, the “crystallization slot,” and they could also be frozen and used to collect structural information. Proteins 2003;50:303–311.

Impaired protein conformational landscapes as revealed in anomalous Arrhenius prefactors

A growing body of data supports a role for protein motion in enzyme catalysis. In particular, the ability of enzymes to sample catalytically relevant conformational substates has been invoked to model kinetic and spectroscopic data. However, direct experimental links between rapidly interconverting conformations and the chemical steps of catalysis remain rare. We report here on the kinetic analysis and characterization of the hydride transfer step catalyzed by a series of mutant thermophilic alcohol dehydrogenases (ht-ADH), presenting evidence for Arrhenius prefactor values that become enormously elevated above an expected value of approximately 1013 s-1 when the enzyme operates below its optimal temperature range. Restoration of normal Arrhenius behavior in the ht-ADH reaction occurs at elevated temperatures. A simple model, in which reduced temperature alters the ability of the ht-ADH variants to sample the catalytically relevant region of conformational space, can reproduce the available data. These findings indicate an impaired landscape that has been generated by the combined condition of reduced temperature and mutation at a single, active-site hydrophobic side chain. The broader implication is that optimal enzyme function requires the maintenance of a relatively smooth landscape that minimizes low energy traps.

Crystal structure of human senescence marker protein 30: insights linking structural, enzymatic, and physiological functions .

Human senescence marker protein 30 (SMP30), which functions enzymatically as a lactonase, hydrolyzes various carbohydrate lactones. The penultimate step in vitamin-C biosynthesis is catalyzed by this enzyme in nonprimate mammals. It has also been implicated as an organophosphate hydrolase, with the ability to hydrolyze diisopropyl phosphofluoridate and other nerve agents. SMP30 was originally identified as an aging marker protein, whose expression decreased androgen independently in aging cells. SMP30 is also referred to as regucalcin and has been suggested to have functions in calcium homeostasis. The crystal structure of the human enzyme has been solved from X-ray diffraction data collected to a resolution of 1.4 A. The protein has a 6-bladed beta-propeller fold, and it contains a single metal ion. Crystal structures have been solved with the metal site bound with either a Ca(2+) or a Zn(2+) atom. The catalytic role of the metal ion has been confirmed by mutagenesis of the metal coordinating residues. Kinetic studies using the substrate gluconolactone showed a k(cat) preference of divalent cations in the order Zn(2+) > Mn(2+) > Ca(2+) > Mg(2+). Notably, the Ca(2+) had a significantly higher value of K(d) compared to those of the other metal ions tested (566, 82, 7, and 0.6 mum for Ca(2+), Mg(2+), Zn(2+), and Mn(2+), respectively), suggesting that the Ca(2+)-bound form may be physiologically relevant for stressed cells with an elevated free calcium level.

Active Site Hydrophobic Residues Impact Hydrogen Tunneling Differently in a Thermophilic Alcohol Dehydrogenase at Optimal versus Nonoptimal Temperatures

A growing body of data suggests that protein motion plays an important role in enzyme catalysis. Two highly conserved hydrophobic active site residues in the cofactor-binding pocket of ht-ADH (Leu176 and V260) have been mutated to a series of hydrophobic side chains of smaller size, as well as one deletion mutant, L176Δ. Mutations decrease k(cat) and increase K(M)(NAD(+)). Most of the observed decreases in effects on k(cat) at pH 7.0 are due to an upward shift in the optimal pH for catalysis; a simple electrostatic model is invoked that relates the change in pK(a) to the distance between the positively charged nicotinamide ring and bound substrate. Structural modeling of the L176Δ and V260A variants indicates the development of a cavity behind the nicotinamide ring without any significant perturbation of the secondary structure of the enzyme relative to that of the wild type. Primary kinetic isotope effects (KIEs) are modestly increased for all mutants. Above the dynamical transition at 30 °C for ht-ADH [Kohen, A., et al. (1999) Nature 399, 496], the temperature dependence of the KIE is seen to increase with a decrease in side chain volume at positions 176 and 260. Additionally, the relative trends in the temperature dependence of the KIE above and below 30 °C appear to be reversed for the cofactor-binding pocket mutants in relation to wild-type protein. The aggregate results are interpreted in the context of a full tunneling model of enzymatic hydride transfer that incorporates both protein conformational sampling (preorganization) and active site optimization of tunneling (reorganization). The reduced temperature dependence of the KIE in the mutants below 30 °C indicates that at low temperatures, the enzyme adopts conformations refractory to donor-acceptor distance sampling.

Human paraoxonase double mutants hydrolyze V and G class organophosphorus nerve agents

Variants of human paraoxonase 1 (PON1) are being developed as catalytic bioscavengers for the organophosphorus chemical warfare agents (OP). It is preferable that the new PON1 variants have broad spectrum hydrolase activities to hydrolyze both G- and V-class OPs. H115W PON1 has shown improvements over wild type PON1 in its capacity to hydrolyze some OP compounds. We improved upon these activities either by substituting a tryptophan (F347W) near the putative active site residues for enhanced substrate binding or by reducing a bulky group (Y71A) at the periphery of the putative enzyme active site. When compared to H115W alone, we found that H115W/Y71A and H115W/F347W maintained VX catalytic efficiency but showed mixed results for the capacity to hydrolyze paraoxon. Testing our double mutants against racemic sarin, we observed reduced values of K(M) for H115W/F347W that modestly improved catalytic efficiency over wild type and H115W. Contrary to previous reports, we show that H115W can hydrolyze soman, and the double mutant H115W/Y71A is nearly 4-fold more efficient than H115W for paraoxon hydrolysis. We also observed modest stereoselectivity for hydrolysis of the P(-) stereoisomer of tabun by H115W/F347W. These data demonstrate enhancements made in PON1 for the purpose of developing an improved catalytic bioscavenger to protect cholinesterase against chemical warfare agents.

The crystal structure of calcium‐ and integrin‐binding protein 1: Insights into redox regulated functions

Calcium‐ and integrin‐binding protein 1 (CIB1) is involved in the process of platelet aggregation by binding the cytoplasmic tail of the αIIb subunit of the platelet‐specific integrin αIibβ3. Although poorly understood, it is widely believed that CIB1 acts as a global signaling regulator because it is expressed in many tissues that do not express integrin αIibβ3. We report the structure of human CIB1 to a resolution of 2.3 Å, crystallized as a dimer. The dimer interface includes an extensive hydrophobic patch in a crystal form with 80% solvent content. Although the dimer form of CIB1 may not be physiologically relevant, this intersub‐unit surface is likely to be linked to αIIb binding and to the binding of other signaling partner proteins. The C‐terminal domain of CIB1 is structurally similar to other EF‐hand proteins such as calmodulin and calcineurin B. Despite structural homology to the C‐terminal domain, the N‐terminal domain of CIB1 lacks calcium‐binding sites. The structure of CIB1 revealed a complex with a molecule of glutathione in the reduced state bond to the N‐terminal domain of one of the two subunits poised to interact with the free thiol of C35. Glutathione bound in this fashion suggests CIB1 may be redox regulated. Next to the bound GSH, the orientation of residues C35, H31, and S48 is suggestive of a cysteine‐type protein phosphatase active site. The potential enzymatic activity of CIB1 is discussed and suggests a mechanism by which it regulates a wide variety of proteins in cells in addition to platelets.

GSTpi modulates JNK activity through a direct interaction with JNK substrate, ATF2

Human GSTpi, an important detoxification enzyme, has been shown to modulate the activity of JNKs by inhibiting apoptosis and by causing cell proliferation and tumor growth. In this work, we describe a detailed analysis of the interaction in vitro between GSTpi and JNK isoforms (both in their inactive and active, phosphorylated forms). The ability of active JNK1 or JNK2 to phosphorylate their substrate, ATF2, is inhibited by two naturally occurring GSTpi haplotypes (Ile105/Ala114, WT or haplotype A, and Val105/Val114, haplotype C). Haplotype C of GSTpi is a more potent inhibitor of JNK activity than haplotype A, yielding 75–80% and 25–45% inhibition, respectively. We show that GSTpi is not a substrate of JNK, as was earlier suggested by others. Through binding studies, we demonstrate that the interaction between GSTpi and phosphorylated, active JNKs is isoform specific, with JNK1 being the preferred isoform. In contrast, GSTpi does not interact with unphosphorylated, inactive JNKs unless a JNK substrate, ATF2, is present. We also demonstrate, for the first time, a direct interaction: between GSTpi and ATF2. GSTpi binds with similar affinity to active JNK + ATF2 and to ATF2 alone. Direct binding experiments between ATF2 and GSTpi, either alone or in the presence of glutathione analogs or phosphorylated ATF2, indicate that the xenobiotic portion of the GSTpi active site and the JNK binding domain of ATF2 are involved in this interaction. Competition between GSTpi and active JNK for the substrate ATF2 may be responsible for the inhibition of JNK catalysis by GSTpi.