John J. Tanner

Redox Regulation of Protein Tyrosine Phosphatases: Structural and Chemical Aspects

Protein tyrosine phosphatases (PTPs) are important targets of the H(2)O(2) that is produced during mammalian signal transduction. H(2)O(2)-mediated inactivation of PTPs also may be important in various pathophysiological conditions involving oxidative stress. Here we review the chemical and structural biology of redox-regulated PTPs. Reactions of H(2)O(2) with PTPs convert the catalytic cysteine thiol to a sulfenic acid. In PTPs, the initially generated sulfenic acid residues have the potential to undergo secondary reactions with a neighboring amide nitrogen or cysteine thiol residue to yield a sulfenyl amide or disulfide, respectively. The chemical mechanisms by which formation of sulfenyl amide and disulfide linkages can protect the catalytic cysteine residue against irreversible overoxidation to sulfinic and sulfonic oxidation states are described. Due to the propensity for back-door and distal cysteine residues to engage with the active-site cysteine after oxidative inactivation, differences in the structures of the oxidatively inactivated PTPs may stem, to a large degree, from differences in the number and location of cysteine residues surrounding the active site of the enzymes. PTPs with key cysteine residues in structurally similar locations may be expected to share similar mechanisms of oxidative inactivation.

Structural Biology of Proline Catabolism

The proline catabolic enzymes proline dehydrogenase and Δ1-pyrroline-5-carboxylate dehydrogenase catalyze the 4-electron oxidation of proline to glutamate. These enzymes play important roles in cellular redox control, superoxide generation, apoptosis and cancer. In some bacteria, the two enzymes are fused into the bifunctional enzyme, proline utilization A. Here we review the three-dimensional structural information that is currently available for proline catabolic enzymes. Crystal structures have been determined for bacterial monofunctional proline dehydrogenase and Δ1-pyrroline-5-carboxylate dehydrogenase, as well as the proline dehydrogenase and DNA-binding domains of proline utilization A. Some of the functional insights provided by analyses of these structures are discussed, including substrate recognition, catalytic mechanism, biochemical basis of inherited proline catabolic disorders and DNA recognition by proline utilization A.

A structurally conserved water molecule in Rossmann dinucleotide-binding domains.

A computational comparison of 102 high‐resolution (≤1.90 Å) enzyme‐dinucleotide (NAD, NADP, FAD) complexes was performed to investigate the role of solvent in dinucleotide recognition by Rossmann fold domains. The typical binding site contains about 9–12 water molecules, and about 30% of the hydrogen bonds between the protein and the dinucleotide are water mediated. Detailed inspection of the structures reveals a structurally conserved water molecule bridging dinucleotides with the well‐known glycine‐rich phosphate‐binding loop. This water molecule displays a conserved hydrogen‐bonding pattern. It forms hydrogen bonds to the dinucleotide pyrophosphate, two of the three conserved glycine residues of the phosphate‐binding loop, and a residue at the C‐terminus of strand four of the Rossmann fold. The conserved water molecule is also present in high‐resolution structures of apo enzymes. However, the water molecule is not present in structures displaying significant deviations from the classic Rossmann fold motif, such as having nonstandard topology, containing a very short phosphate‐binding loop, or having α‐helix “A” oriented perpendicular to the β‐sheet. Thus, the conserved water molecule appears to be an inherent structural feature of the classic Rossmann dinucleotide‐binding domain.

Structure of the proline dehydrogenase domain of the multifunctional PutA flavoprotein

The PutA flavoprotein from Escherichia coli plays multiple roles in proline catabolism by functioning as a membrane-associated bi-functional enzyme and a transcriptional repressor of proline utilization genes. The human homolog of the PutA proline dehydrogenase (PRODH) domain is critical in p53-mediated apoptosis and schizophrenia. Here we report the crystal structure of a 669-residue truncated form of PutA that shows both PRODH and DNA-binding activities, representing the first structure of a PutA protein and a PRODH enzyme from any organism. The structure is a domain-swapped dimer with each subunit comprising three domains: a helical dimerization arm, a 120-residue domain containing a three-helix bundle similar to that in the helix-turn-helix superfamily of DNA-binding proteins and a β/α-barrel PRODH domain with a bound lactate inhibitor. Analysis of the structure provides insight into the mechanism of proline oxidation to pyrroline-5-carboxylate, and functional studies of a mutant protein suggest that the DNA-binding domain is located within the N-terminal 261 residues of E. coli PutA.

Structure and Kinetics of Monofunctional Proline Dehydrogenase from Thermus thermophilus.

Proline dehydrogenase (PRODH) and Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDH) catalyze the two-step oxidation of proline to glutamate. They are distinct monofunctional enzymes in all eukaryotes and some bacteria but are fused into bifunctional enzymes known as proline utilization A (PutA) in other bacteria. Here we report the first structure and biochemical data for a monofunctional PRODH. The 2.0-Å resolution structure of Thermus thermophilus PRODH reveals a distorted (βα)8 barrel catalytic core domain and a hydrophobic α-helical domain located above the carboxyl-terminal ends of the strands of the barrel. Although the catalytic core is similar to that of the PutA PRODH domain, the FAD conformation of T. thermophilus PRODH is remarkably different and likely reflects unique requirements for membrane association and communication with P5CDH. Also, the FAD of T. thermophilus PRODH is highly solvent-exposed compared with PutA due to a 4-Å shift of helix 8. Structure-based sequence analysis of the PutA/PRODH family led us to identify nine conserved motifs involved in cofactor and substrate recognition. Biochemical studies show that the midpoint potential of the FAD is -75 mV and the kinetic parameters for proline are Km = 27 mm and kcat = 13 s-1. 3,4-Dehydro-l-proline was found to be an efficient substrate, and l-tetrahydro-2-furoic acid is a competitive inhibitor (KI = 1.0 mm). Finally, we demonstrate that T. thermophilus PRODH reacts with O2 producing superoxide. This is significant because superoxide production underlies the role of human PRODH in p53-mediated apoptosis, implying commonalities between eukaryotic and bacterial monofunctional PRODHs.

High-resolution structure of human D-glyceraldehyde-3-phosphate dehydrogenase.

GAPDH (D-glyceraldehyde-3-phosphate dehydrogenase) is a multifunctional protein that is a target for the design of antitrypanosomatid and anti-apoptosis drugs. Here, the first high-resolution (1.75 Angstroms) structure of a human GAPDH is reported. The structure shows that the intersubunit selectivity cleft that has been leveraged in the design of antitrypanosomatid compounds is closed in human GAPDH. Modeling of an anti-trypanosomatid GAPDH inhibitor in the human GAPDH active site provides insights into the basis for the observed selectivity of this class of inhibitor. Moreover, the high-resolution data reveal a new feature of the cleft: water-mediated intersubunit hydrogen bonds that assist closure of the cleft in the human enzyme. The structure is used in a computational ligand-docking study of the small-molecule compound CGP-3466, which inhibits apoptosis by preventing nuclear accumulation of GAPDH. Plausible binding sites are identified in the adenosine pocket of the NAD(+)-binding site and in a hydrophobic channel located in the center of the tetramer near the intersection of the three molecular twofold axes. The structure is also used to build a qualitative model of the complex between GAPDH and the E3 ubiquitin ligase Siah1. The model suggests that the convex surface near GAPDH Lys227 interacts with a large shallow groove of the Siah1 dimer. These results are discussed in the context of the recently discovered NO-S-nitrosylation-GAPDH-Siah1 apoptosis cascade.

X-ray crystal structures of native HIV-1 capsid protein reveal conformational variability

Retroviral capsids in their native form Capsid proteins of retroviruses form protective lattices around viral RNA molecules. The precise molecular details of how individual, full-length capsid proteins assemble to shield the viral genome; however, are not well understood. Obal et al. and Gres et al. now report high resolution crystal structures of the full length capsid proteins from Bovine Leukemia Virus and HIV-1, respectively. The two studies complement each other to reveal the dynamic nature of capsid protein assembly and of how individual capsid proteins interact in the lattice. The findings may have relevance for drug design. Science, this issue p. 95; see also p. 99 Crystal structures of native retroviral capsid proteins reveal how these large protein structures assemble and interact. The detailed molecular interactions between native HIV-1 capsid protein (CA) hexamers that shield the viral genome and proteins have been elusive. We report crystal structures describing interactions between CA monomers related by sixfold symmetry within hexamers (intrahexamer) and threefold and twofold symmetry between neighboring hexamers (interhexamer). The structures describe how CA builds hexagonal lattices, the foundation of mature capsids. Lattice structure depends on an adaptable hydration layer modulating interactions among CA molecules. Disruption of this layer alters interhexamer interfaces, highlighting an inherent structural variability. A CA-targeting antiviral affects capsid stability by binding across CA molecules and subtly altering interhexamer interfaces remote to the ligand-binding site. Inherent structural plasticity, hydration layer rearrangement, and effector binding affect capsid stability and have functional implications for the retroviral life cycle.

Ab initio study of proton transfer in [H3N-H-NH3]+ and [H3N-H-OH2]+

Abstract Quantum mechanical ab initio calculations at the MP2/6-31G * level are performed on two proton bound dimer systems, [H 3 N−H−NH 3 ] + and [H 3 N−H−OH 2 ] + . Several calculations using a medium-size polarized basis set were performed as a check of the 6-31G * results. Energies are calculated at heavy-atom separations of 2.25–3.25 A. At fixed monomer separations, H is moved along the intermonomer aixs, thus mapping out the proton transfer potential energy surface. For the ammonia dimer, the energy for displacements of H perpendicular to the N—N axis are also calculated. For the ammonia—water dimer, two different binding geometries for the water molecule are considered. All data are fit to analytical functions. We discuss the effects of squeezing and stretching the donor—acceptor distance on proton transfer.

A comparative study of time dependent quantum mechanical wave packet evolution methods

We present a detailed comparison of the efficiency and accuracy of the second‐ and third‐order split operator methods, a time dependent modified Cayley method, and the Chebychev polynomial expansion method for solving the time dependent Schrodinger equation in the one‐dimensional double well potential energy function. We also examine the efficiency and accuracy of the split operator and modified Cayley methods for the imaginary time propagation.

Crystal structure of the bifunctional proline utilization A flavoenzyme from Bradyrhizobium japonicum

The bifunctional proline catabolic flavoenzyme, proline utilization A (PutA), catalyzes the oxidation of proline to glutamate via the sequential activities of FAD-dependent proline dehydrogenase (PRODH) and NAD+-dependent Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDH) domains. Although structures for some of the domains of PutA are known, a structure for the full-length protein has not previously been solved. Here we report the 2.1 Å resolution crystal structure of PutA from Bradyrhizobium japonicum, along with data from small-angle x-ray scattering, analytical ultracentrifugation, and steady-state and rapid-reaction kinetics. PutA forms a ring-shaped tetramer in solution having a diameter of 150 Å. Within each protomer, the PRODH and P5CDH active sites face each other at a distance of 41 Å and are connected by a large, irregularly shaped cavity. Kinetics measurements show that glutamate production occurs without a lag phase, suggesting that the intermediate, Δ1-pyrroline-5-carboxylate, is preferably transferred to the P5CDH domain rather than released into the bulk medium. The structural and kinetic data imply that the cavity serves both as a microscopic vessel for the hydrolysis of Δ1-pyrroline-5-carboxylate to glutamate semialdehyde and a protected conduit for the transport of glutamate semialdehyde to the P5CDH active site.