The detailed molecular interactions between Human Immunodeficiency Virus type 1 (HIV-1) capsid protein (CA) hexamers have been elusive in the context of a native protein. We report crystal structures describing novel interactions between CA monomers related by 6-fold symmetry within a hexamer (intra-hexamer) and by 3-fold and 2-fold symmetry between neighboring hexamers (inter-hexamer). These structures help elucidate how CA builds a hexagonal lattice, the foundation of the mature capsid. Lattice structure depends on an adaptable hydration layer that modulates interactions among CA molecules. Disruption of this layer by crystal dehydration treatment alters inter-hexamer interfaces and condenses CA packing, highlighting an inherent structural variability. Capsid stability changes imparted by high concentrations of CA-targeting antiviral PF74 can be explained by variations at inter-hexamer interfaces remote to the ligand binding site. Inherent structural plasticity, hydration layer rearrangement, and effector molecule binding may perturb capsid uncoating or assembly and have functional implications for the retroviral life cycle.

The proline catabolic enzymes proline dehydrogenase and Delta(1)-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 Delta(1)-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.

Proline dehydrogenase (PRODH) and ⌬ 1 -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 (␤␣) 8 barrel catalytic core domain and a hydrophobic ␣-helical domain located above the carboxylterminal 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. Structurebased 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 K m ‫؍‬ 27 mM and k cat ‫؍‬ 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 (K I ‫؍‬ 1.0 mM). Finally, we demonstrate that T. thermophilus PRODH reacts with O 2 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.Oxidation of amino acids is a central part of energy metabolism. The oxidative pathway for proline consists of two enzymatic steps and an intervening nonenzymatic equilibrium (Scheme 1) (1, 2). The first enzymatic step transforms proline to ⌬ 1 -pyrroline-5-carboxylate (P5C), 2 which is non-enzymatically hydrolyzed to glutamic semialdehyde. The semialdehyde is oxidized in the second enzymatic step to glutamate.This 4-electron transformation of proline is common to all organisms, but the enzymes of proline catabolism differ widely among the three kingdoms of life. Amino acid sequence analysis shows that bacteria and eukaryotes share a common set of proline catabolic enzymes called proline dehydrogenase (PRODH) and P5C dehydrogenase (P5CDH). Studies of the bacterial enzymes have shown that PRODH is an FAD-dependent enzyme with a (␤␣) 8 barrel catalytic core (3, 4), and P5CDH is an NAD ϩ -dependent Rossmann fold enzyme featuring a nucleophilic Cys (5). These enzymes are unrelated in sequence and structure to hyperthermophilic archaeal proline catabolic enzymes, which appear in unique hetero-tetrameric and hetero-octameric complexes (6).An intriguing aspect of proline catabolism in eukaryotes and bacteria is that PRODH and P5CDH are separate enzymes in some organisms, whereas the two enzymes are fused in other organisms. The traditional view has been that P...

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 beta/alpha-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.

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.