Discovery of Hydroxylase Activity for PqqB Provides a Missing Link in the Pyrroloquinoline Quinone Biosynthetic Pathway.

Understanding the biosynthesis of cofactors is fundamental to the life sciences, yet to date a few important pathways remain unresolved. One example is the redox cofactor pyrroloquinoline quinone (PQQ), which is critical for C1 metabolism in many microorganisms, a disproportionate number of which are opportunistic human pathogens. While the initial and final steps of PQQ biosynthesis, involving PqqD/E and PqqC, have been elucidated, the precise nature and order of the remaining transformations in the pathway are unknown. Here we show evidence that the remaining essential biosynthetic enzyme PqqB is an iron-dependent hydroxylase catalyzing oxygen-insertion reactions that are proposed to produce the quinone moiety of the mature PQQ cofactor. The demonstrated reactions of PqqB are unprecedented within the metallo ß-lactamase protein family and expand the catalytic repertoire of nonheme iron hydroxylases. These new findings also generate a nearly complete description of the PQQ biosynthetic pathway.

Crystal structures reveal metal-binding plasticity at the metallo-ß-lactamase active site of PqqB from Pseudomonas putida.

PqqB is an enzyme involved in the biosynthesis of pyrroloquinoline quinone and a distal member of the metallo-ß-lactamase (MBL) superfamily. PqqB lacks two residues in the conserved signature motif HxHxDH that makes up the key metal-chelating elements that can bind up to two metal ions at the active site of MBLs and other members of its superfamily. Here, we report crystal structures of PqqB bound to Mn2+, Mg2+, Cu2+, and Zn2+. These structures demonstrate that PqqB can still bind metal ions at the canonical MBL active site. The fact that PqqB can adapt its side chains to chelate a wide spectrum of metal ions with different coordination features on a uniform main chain scaffold demonstrates its metal-binding plasticity. This plasticity may provide insights into the structural basis of promiscuous activities found in ensembles of metal complexes within this superfamily. Furthermore, PqqB belongs to a small subclass of MBLs that contain an additional CxCxxC motif that binds a structural Zn2+. Our data support a key role for this motif in dimerization.

Human Rhinovirus Presenting 4E10 Epitope of HIV-1 MPER Elicits Neutralizing Antibodies in Human ICAM-1 Transgenic Mice.

Attempts at eliciting neutralizing antibodies against human immunodeficiency virus (HIV)-1 have generally failed. Computationally designed epitope-scaffold platforms allow transplantation of structural epitopes to scaffold proteins. Human rhinovirus (HRV) allows such engrafting of HIV-1 epitopes on the surface scaffold proteins. However, since HRV infects only humans and great apes, the efficacy of chimeric HRV-based live viral vaccines is difficult to assess in animal models. Here, we used human ICAM-1 transgenic (hICAM-1 Tg) mice that support productive HRV infection to assess the efficacy of chimeric HRV expressing the HIV-1 membrane proximal external region (MPER) epitope, 4E10. Intranasal immunization with chimeric HRV in transgenic mice effectively induced antibodies that recognized 4E10 peptide as well as HIV-1 Env trimer. Importantly, the immunized mouse sera were able to neutralize HIV strains including those belonging to clades B and C. Moreover, intranasal immunization could bypass pre-existing immunity to HRV. Thus, chimeric HRV appears to provide a viable vaccine vehicle for HIV-1 immunization in humans.

The macular degeneration-linked C1QTNF5 (S163) mutation causes higher-order structural rearrangements.

The C1q-tumor necrosis factor 5 (C1QTNF5) protein plays a significant role in retinal pigmented epithelium (RPE) cellular adhesion. The C1QTNF5 gene is co-transcribed with the frizzled-related protein (MFRP) gene. A Ser-to-Arg mutation at site 163 (S163R) in C1QTNF5 is known to cause late-onset retinal macular degeneration (L-ORMD). Here we also found that C1QTNF5 monomers can multimerize into a bouquet-like octadecamer. We found that a novel intermolecular hydrogen-bond network of S163 that glues adjacent globular heads of C1QTNF5 together was weakened or abolished by the R163 pathogenic mutation. These findings could underlie the structural basis of this protein's adhesive function and relate to the pathogenesis of its S163R mutation. Additionally, the fact that C1QTNF5 immobilized to a resin selectively enriched detergent extracted membrane-bound MFRP, further confirmed their interaction, implying functions other than cellular adhesion for C1QTNF5.

Crystal structure of the globular domain of C1QTNF5: Implications for late-onset retinal macular degeneration.

Autosomal dominant late-onset retinal macular degeneration (L-ORMD) is caused by a single S163R mutation in the C1q and tumor necrosis factor-related protein 5 (C1QTNF5) gene. The C1QTNF5 gene encodes a secreted and membrane-associated protein involved in adhesion of retinal pigmented epithelial cells (RPE) to Bruch's membrane. The crystal structure of the trimeric globular domain of human C1QTNF5 at 1.34Å resolution reveals unique features of this novel C1q family member. It lacks a Ca²âº-binding site, displays a remarkable non-uniform distribution of surface electrostatic potentials and possesses a unique sequence (F181F182G183G184W185P186) that forms a hydrophobic plateau surrounded by Lys and Arg residues with a solvent cavity underneath. S163 forms a hydrogen bond with F182 in a hydrophobic area extending to the hydrophobic plateau. The pathogenic mutation S163R disrupts this hydrogen bonding and positively charges these hydrophobic areas. Thus, our analysis provides insights into the structural basis of the L-ORMD disease mechanism.

HIV-1 reverse transcriptase (RT) polymorphism 172K suppresses the effect of clinically relevant drug resistance mutations to both nucleoside and non-nucleoside RT inhibitors.

Polymorphisms have poorly understood effects on drug susceptibility and may affect the outcome of HIV treatment. We have discovered that an HIV-1 reverse transcriptase (RT) polymorphism (RT(172K)) is present in clinical samples and in widely used laboratory strains (BH10), and it profoundly affects HIV-1 susceptibility to both nucleoside (NRTIs) and non-nucleoside RT inhibitors (NNRTIs) when combined with certain mutations. Polymorphism 172K significantly suppressed zidovudine resistance caused by excision (e.g. thymidine-associated mutations) and not by discrimination mechanism mutations (e.g. Q151M complex). Moreover, it attenuated resistance to nevirapine or efavirenz imparted by NNRTI mutations. Although 172K favored RT-DNA binding at an excisable pre-translocation conformation, it decreased excision by thymidine-associated mutation-containing RT. 172K affected DNA handling and decreased RT processivity without significantly affecting the k(cat)/K(m) values for dNTP. Surface plasmon resonance experiments revealed that RT(172K) decreased DNA binding by increasing the dissociation rate. Hence, the increased zidovudine susceptibility of RT(172K) results from its increased dissociation from the chain-terminated DNA and reduced primer unblocking. We solved a high resolution (2.15 Å) crystal structure of RT mutated at 172 and compared crystal structures of RT(172R) and RT(172K) bound to NNRTIs or DNA/dNTP. Our structural analyses highlight differences in the interactions between α-helix E (where 172 resides) and the active site ß9-strand that involve the YMDD loop and the NNRTI binding pocket. Such changes may increase dissociation of DNA, thus suppressing excision-based NRTI resistance and also offset the effect of NNRTI resistance mutations thereby restoring NNRTI binding.

Structural basis of HIV-1 resistance to AZT by excision.

Human immunodeficiency virus (HIV-1) develops resistance to 3'-azido-2',3'-deoxythymidine (AZT, zidovudine) by acquiring mutations in reverse transcriptase that enhance the ATP-mediated excision of AZT monophosphate from the 3' end of the primer. The excision reaction occurs at the dNTP-binding site, uses ATP as a pyrophosphate donor, unblocks the primer terminus and allows reverse transcriptase to continue viral DNA synthesis. The excision product is AZT adenosine dinucleoside tetraphosphate (AZTppppA). We determined five crystal structures: wild-type reverse transcriptase-double-stranded DNA (RT-dsDNA)-AZTppppA; AZT-resistant (AZTr; M41L D67N K70R T215Y K219Q) RT-dsDNA-AZTppppA; AZTr RT-dsDNA terminated with AZT at dNTP- and primer-binding sites; and AZTr apo reverse transcriptase. The AMP part of AZTppppA bound differently to wild-type and AZTr reverse transcriptases, whereas the AZT triphosphate part bound the two enzymes similarly. Thus, the resistance mutations create a high-affinity ATP-binding site. The structure of the site provides an opportunity to design inhibitors of AZT-monophosphate excision.

Structural basis for the role of the K65R mutation in HIV-1 reverse transcriptase polymerization, excision antagonism, and tenofovir resistance.

K65R is a primary reverse transcriptase (RT) mutation selected in human immunodeficiency virus type 1-infected patients taking antiretroviral regimens containing tenofovir disoproxil fumarate or other nucleoside analog RT drugs. We determined the crystal structures of K65R mutant RT cross-linked to double-stranded DNA and in complexes with tenofovir diphosphate (TFV-DP) or dATP. The crystals permit substitution of TFV-DP with dATP at the dNTP-binding site. The guanidinium planes of the arginines K65R and Arg(72) were stacked to form a molecular platform that restricts the conformational adaptability of both of the residues, which explains the negative effects of the K65R mutation on nucleotide incorporation and on excision. Furthermore, the guanidinium planes of K65R and Arg(72) were stacked in two different rotameric conformations in TFV-DP- and dATP-bound structures that may help explain how K65R RT discriminates the drug from substrates. These K65R-mediated effects on RT structure and function help us to visualize the complex interaction with other key nucleotide RT drug resistance mutations, such as M184V, L74V, and thymidine analog resistance mutations.

The atomic resolution crystal structure of atratoxin determined by single wavelength anomalous diffraction phasing.

By using single wavelength anomalous diffraction phasing based on the anomalous signal from copper atoms, the crystal structure of atratoxin was determined at the resolution of 1.5 A and was refined to an ultrahigh resolution of 0.87 A. The ultrahigh resolution electron density maps allowed the modeling of 38 amino acid residues in alternate conformations and the location of 322 of 870 possible hydrogen atoms. To get accurate information at the atomic level, atratoxin-b (an analog of atratoxin with reduced toxicity) was also refined to an atomic resolution of 0.92 A. By the sequence and structural comparison of these two atratoxins, Arg(33) and Arg(36) were identified to be critical to their varied toxicity. The effect of copper ions on the distribution of hydrogen atoms in atratoxin was discussed, and the interactions between copper ions and protein residues were analyzed based on a statistical method, revealing a novel pentahedral copper-binding motif.

Purification, partial characterization, crystallization and preliminary X-ray diffraction of two cysteine-rich secretory proteins from Naja atra and Trimeresurus stejnegeri venoms.

Cysteine-rich secretory proteins (CRISPs) are widely distributed in mammals and snake venoms. They possess apparent homology but varying functions. The structure of CRISPs has remained elusive. Two novel members of the family, natrin and stecrisp, have been purified from Naja atra and Trimeresurus stejnegeri venoms, respectively. Their crystals diffract X-rays to resolution limits of 2.1 and 1.6 angstroms, respectively, and belong to the orthorhombic system with different space groups, unit-cell parameters and numbers of molecules per asymmetric unit. Their structures will contribute a structural basis for further functional studies of this family.

Purification, N-terminal sequencing, crystallization and preliminary structural determination of atratoxin-b, a short-chain alpha-neurotoxin from Naja atra venom.

Atratoxin-b, a short-chain alpha-neurotoxin purified from Naja atra (mainland Chinese cobra) venom using a three-step chromatography procedure, has an apparent molecular mass of 6950 Da with an alkaline pI value (>9.5) and consists of one single polypeptide chain as estimated by MALDI-TOF mass spectrometry and SDS-PAGE. The protein is toxic to mice, with an in vitro LD(50) of about 0.18 mg kg(-1). Its N-terminal amino-acid sequence, LECHNQQSSQTPTIT, displays a very high homology to those of other alpha-neurotoxins. The overall three-dimensional structure of atratoxin-b is very similar to that of the homologous erabutoxin-a, as shown by the crystallographic molecular replacement and preliminary refinement results, with an R factor and R(free) of 27 and 29%, respectively. The microcrystal slowly grew to dimensions of approximate 0.1 x 0.1 x 0.15 mm over eight months using hanging-drop vapour-diffusion method. It gave a set of diffraction data to 1.56 A resolution using X-rays of wavelength 1.1516 A generated by the X-ray Diffraction and Scattering Station of beamline U7B at the National Synchrotron Radiation Laboratory (Hefei, China); this is the first example of the use of this beamline in protein crystallography. The crystals belong to the tetragonal space group P4(1)2(1)2, with unit-cell parameters a = 49.28, c = 44.80 A, corresponding to one molecule per asymmetric unit and a volume-to-mass ratio of 1.96 A(3) Da(-1).

Purification, N-terminal sequencing, crystallization and preliminary X-ray diffraction analysis of atratoxin, a new short-chain alpha-neurotoxin from the venom of Naja naja atra.

Atratoxin, a new alpha-neurotoxin purified to homogeneity by a series of liquid chromatographies from the venom of Naja naja atra (mainland Chinese cobra), is a small single-polypeptide alkaline protein with a pI of about 9.5 and molecular weight of 6952 Da estimated by mass spectrometry. Although the sequencing of the N-terminal 15 residues (LECHNQQTTQQPEGG) shows that this neurotoxic protein contains most of the residues, especially at the conserved positions, of the consensus sequence of short-chain alpha-neurotoxins, the natural mutations in the N-terminal Loop-1 presented by the sequence alignment may have structural or functional implications for the interactions between alpha-neurotoxins and related receptors. Single crystals of atratoxin have been grown from drops containing the necessary Cu(2+) ions by the conventional hanging-drop vapour-diffusion method. The crystals diffract X-rays to 1.6 A resolution and belong to space group C222(1), with unit-cell parameters a = 47.36, b = 47.83, c = 91.31 A, corresponding to a volume-to-mass ratio of 1.85 A(3) Da(-1) and two molecules in each asymmetric unit.