The initial step in target cell infection by human, and the closely related simian immunodeficiency viruses (HIV and SIV, respectively) occurs with the binding of trimeric envelope glycoproteins (Env), composed of heterodimers of the viral transmembrane glycoprotein (gp41) and surface glycoprotein (gp120) to target T-cells. Knowledge of the molecular structure of trimeric Env on intact viruses is important both for understanding the molecular mechanisms underlying virus-cell interactions and for the design of effective immunogen-based vaccines to combat HIV/AIDS. Previous analyses of intact HIV-1 BaL virions have already resulted in structures of trimeric Env in unliganded and CD4-liganded states at ∼20 Å resolution. Here, we show that the molecular architectures of trimeric Env from SIVmneE11S, SIVmac239 and HIV-1 R3A strains are closely comparable to that previously determined for HIV-1 BaL, with the V1 and V2 variable loops located at the apex of the spike, close to the contact zone between virus and cell. The location of the V1/V2 loops in trimeric Env was definitively confirmed by structural analysis of HIV-1 R3A virions engineered to express Env with deletion of these loops. Strikingly, in SIV CP-MAC, a CD4-independent strain, trimeric Env is in a constitutively “open” conformation with gp120 trimers splayed out in a conformation similar to that seen for HIV-1 BaL Env when it is complexed with sCD4 and the CD4i antibody 17b. Our findings suggest a structural explanation for the molecular mechanism of CD4-independent viral entry and further establish that cryo-electron tomography can be used to discover distinct, functionally relevant quaternary structures of Env displayed on intact viruses.

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 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 þ -dependent Δ 1 -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, Δ 1 -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 Δ 1 -pyrroline-5-carboxylate to glutamate semialdehyde and a protected conduit for the transport of glutamate semialdehyde to the P5CDH active site.proline catabolism | substrate channeling

Adeno-Associated Virus is the leading vector for gene therapy. Although it is the vector for all in vivo gene therapies approved for clinical use by the US Food and Drug Administration, its biology is still not yet fully understood. It has been shown that different serotypes of AAV bind to their cellular receptor, AAVR, in different ways. Previously we have reported a 2.4Å structure of AAV2 bound to AAVR that shows ordered structure for only one of the two AAVR domains with which AAV2 interacts. In this study we present a 2.5Å resolution structure of AAV5 bound to AAVR. AAV5 binds to the first polycystic kidney disease (PKD) domain of AAVR that was not ordered in the AAV2 structure. Interactions of AAV5 with AAVR are analyzed in detail, and the implications for AAV2 binding are explored through molecular modeling. Moreover, we find that binding sites for the antibodies ADK5a, ADK5b, and 3C5 on AAV5 overlap with the binding site of AAVR. These insights provide a structural foundation for development of gene therapy agents to better evade immune neutralization without disrupting cellular entry.