The pyrroloquinoline quinone biosynthesis pathway revisited: A structural approach

<p>Abstract</p> <p>Background</p> <p>The biosynthesis pathway of Pyrroloquinoline quinone, a bacterial redox active cofactor for numerous alcohol and aldose dehydrogenases, is largely unknown, but it is proven that at least six genes in <it>Klebsiella pneumoniae </it>(PqqA-F) are required, all of which are located in the PQQ-operon.</p> <p>Results</p> <p>New structural data of some PQQ biosynthesis proteins and their homologues provide new insights and functional assignments of the proteins in the pathway. Based on sequence analysis and homology models we propose the role and catalytic function for each enzyme involved in this intriguing biosynthesis pathway.</p> <p>Conclusion</p> <p>PQQ is derived from the two amino acids glutamate and tyrosine encoded in the precursor peptide PqqA. Five reactions are necessary to form this quinone cofactor. The PqqA peptide is recognised by PqqE, which links the C9 and C9a, afterwards it is accepted by PqqF which cuts out the linked amino acids. The next reaction (Schiff base) is spontaneous, the following dioxygenation is catalysed by an unknown enzyme. The last cyclization and oxidation steps are catalysed by PqqC. Taken together the known facts of the different proteins we assign a putative function to all six proteins in PQQ biosynthesis pathway.</p

Purification, crystallization and X-ray diffraction analysis of human dynamin-related protein 1 GTPase-GED fusion protein

The mechano-enzyme dynamin-related protein 1 plays an important role in mitochondrial fission and is implicated in cell physiology. Dysregulation of Drp1 is associated with abnormal mitochondrial dynamics and neuronal damage. Drp1 shares structural and functional similarities with dynamin 1 with respect to domain organization, ability to self-assemble into spiral-like oligomers and GTP-cycle-dependent membrane scission. Structural studies of human dynamin-1 have greatly improved the understanding of this prototypical member of the dynamin superfamily. However, high-resolution structural information for full-length human Drp1 covering the GTPase domain, the middle domain and the GTPase effector domain (GED) is still lacking. In order to obtain mechanistic insights into the catalytic activity, a nucleotide-free GTPase-GED fusion protein of human Drp1 was expressed, purified and crystallized. Initial X-ray diffraction experiments yielded data to 2.67 angstrom resolution. The hexagonal-shaped crystals belonged to space group P2(1)2(1)2, with unit-cell parameters a = 53.59, b = 151.65, c = 43.53 angstrom, one molecule per asymmetric unit and a solvent content of 42%. Expression of selenomethionine-labelled protein is currently in progress. Here, the expression, purification, crystallization and X-ray diffraction analysis of the Drp1 GTPase-GED fusion protein are presented, which form a basis for more detailed structural and biophysical analysis

Functional mapping of human dynamin-1-like GTPase domain based on x-ray structure analyses.

Human dynamin-1-like protein (DNM1L) is a GTP-driven molecular machine that segregates mitochondria and peroxisomes. To obtain insights into its catalytic mechanism, we determined crystal structures of a construct comprising the GTPase domain and the bundle signaling element (BSE) in the nucleotide-free and GTP-analogue-bound states. The GTPase domain of DNM1L is structurally related to that of dynamin and binds the nucleotide 5'-Guanylyl-imidodiphosphate (GMP-PNP) via five highly conserved motifs, whereas the BSE folds into a pocket at the opposite side. Based on these structures, the GTPase center was systematically mapped by alanine mutagenesis and kinetic measurements. Thus, residues essential for the GTPase reaction were characterized, among them Lys38, Ser39 and Ser40 in the phosphate binding loop, Thr59 from switch I, Asp146 and Gly149 from switch II, Lys216 and Asp218 in the G4 element, as well as Asn246 in the G5 element. Also, mutated Glu81 and Glu82 in the unique 16-residue insertion of DNM1L influence the activity significantly. Mutations of Gln34, Ser35, and Asp190 in the predicted assembly interface interfered with dimerization of the GTPase domain induced by a transition state analogue and led to a loss of the lipid-stimulated GTPase activity. Our data point to related catalytic mechanisms of DNM1L and dynamin involving dimerization of their GTPase domains

Structural and functional analysis of the NLRP4 pyrin domain

NLRP4 is a member of the nucleotide-binding and leucine-rich repeat receptor (NLR) family of cytosolic receptors and a member of an inflammation signaling cascade. Here, we present the crystal structure of the NLRP4 pyrin domain (PYD) at 2.3 Å resolution. The NLRP4 PYD is a member of the death domain (DD) superfamily and adopts a DD fold consisting of six α-helices tightly packed around a hydrophobic core, with a highly charged surface that is typical of PYDs. Importantly, however, we identified several differences between the NLRP4 PYD crystal structure and other PYD structures that are significant enough to affect NLRP4 function and its interactions with binding partners. Notably, the length of helix α3 and the α2−α3 connecting loop in the NLRP4 PYD are unique among PYDs. The apoptosis-associated speck-like protein containing a CARD (ASC) is an adaptor protein whose interactions with a number of distinct PYDs are believed to be critical for activation of the inflammatory response. Here, we use co-immunoprecipitation, yeast two-hybrid, and nuclear magnetic resonance chemical shift perturbation analysis to demonstrate that, despite being important for activation of the inflammatory response and sharing several similarities with other known ASC-interacting PYDs (i.e., ASC2), NLRP4 does not interact with the adaptor protein ASC. Thus, we propose that the factors governing homotypic PYD interactions are more complex than the currently accepted model, which states that complementary charged surfaces are the main determinants of PYD–PYD interaction specificity

Data collection and processing statistics.

<p>Both crystals belong to the orthorhombic space group P2₁2₁2 with two 2-fold screw axes (a, b) and a 2-fold axis (c), all angles being 90°. X-ray diffraction reflections were observed with a redundancy (multiplicity) in the range of 3.5 to 4.1, which improved the data quality by averaging observations. In both cases, the overall signal-to-noise ratio I/σ was around 8, reaching its limit of around 2 at the maximum resolution of 2.3 Ångström. The redundancy-dependent factor R_merge, the redundancy-independent factor R_meas (R_rim), and the precision indicating R_pim were calculated as deviations from averaged reflection intensities (I) according to the given formulas and indicate the data quality. Solvent content refers to the volume of disordered aqueous buffer within the protein crystal lattice.</p>*<p>alues in parentheses refer to the highest resolution shell.</p>a<p>The redundancy dependent merging R-factor: .</p>b<p>The redundancy independent R-factor: .</p>c<p>The precision indicating merging R-factor: .</p>d<p>Mean (I/sd(I)) from SCALA.</p

Superposition of the two DNM1L GG structures and dynamin-1 GG.

<p>(<b>A</b>) Overlay of the nucleotide-free DNM1L GG structure in white with the GMP-PNP-bound structure in green (shown without ligands). Side chains that were mutated in our study are shown as stick models with sequence number labels. (<b>B</b>) Overlay of dynamin-1 (PDB code 2X2F) in yellow with the structure of GMP-PNP-bound DNM1L in green. Mutated residues of DNM1L that are equivalent to those of dynamin (see Fig. 3A) are displayed as side chain stick models with dynamin sequence numbers (depicted without ligands).</p

GTPase activity of DNM1L and the mutants.

<p>(<b>A</b>) Basal GTPase activities of wild-type DNM1L, DNM1L GG fusion protein and full-length mutants. Steady-state GTPase activities of full-length wild-type DNM1L, GG fusion protein, active site mutants and predicted GTPase domain dimerization mutants (Q34A, S35A, D190A) were measured as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071835#s2" target="_blank">Methods</a> section. Amino acid substitutions Q34A, K38A, S39A, T59A, D146A, G149A, K216A and D218A completely abolished GTP hydrolysis. The Q34A mutant is shown as one representative example for the inactive mutants. Among all these mutants, only S35A, S40A, D190A and N246A exhibited significant GTPase turnover. For S35A, both the simple Michaelis-Menten equation fit (label MM, orange dots) and the curve using a cooperative model (continuous orange line) with a Hill coefficient of 2.2 are depicted. Data are means of at least three independent experiments ± standard deviation (displayed as error bars) evaluated by nonlinear regression analysis. (<b>B</b>) Liposome-stimulated GTP hydrolysis of DNM1L and its mutants determined by multiple-turnover assays. Reactions were performed for 12 min at 37°C in the absence (grey bars) or presence (black bars) of PS liposomes. Initial hydrolysis rates k_obs were determined by applying a linear fit to the data, with bars representing mean value ± standard deviation of three independent experiments. For mutants Q34A, S39A, T59A, D146A, G149A, and K216A less then 4% of the GTP was hydrolyzed within 12 minutes. (<b>C</b>) Basal GTP activity of full length DNM1L and the two loop mutants E81A and E81A/E82A. Although the three variants exhibit similar Michaelis-Menten curves, both mutants displayed lower V_max (k_obs) and faster saturation with GTP compared to WT.</p

GTPase domain interface model of the DNM1L GG fusion protein and nucleotide-dependent dimerization.

<p>(<b>A</b>) Two chains of DNM1L molecules were superimposed on the GTPase domain dimer of <i>At</i>Drp1A (PDB code 3T34) as molecules A (green) and B (orange). The interface connecting residues Gln34, Ser35, Asp190, and GTP are depicted as stick models. In addition, the movement of the BSE domains between the pre- and postfission states is represented by the extended <i>At</i>Drp1A dimer (white) and the compact DNM1L dimer. The tetramer model (bottom, left) is based on full-length dynamin-1, which may further oligomerize via the stalks and other GTPase domains (green, orange). (<b>B</b>) Close-up view of the interface at Asp190 from molecule B and Gln34, Ser35 and GTP from molecule A. The conformations of the nucleotide-free and GMP-PNP bound structures are displayed. (<b>C</b>) Dimerization ability of the DNM1L GG fusion protein in the presence of different nucleotides. The GG fusion protein (60 µM) was subjected to gel filtration after incubation with different guanine nucleotide analogs (2 mM). Protein standards at 29 and 75 kDa are indicated. The dimeric protein eluted at a retention volume of 9.5 ml and monomeric protein at 11 ml. (<b>D</b>) SDS PAGE analysis of the SEC runs. Lane 1 shows purified GG fusion protein (41 kDa) followed by a molecular weight protein ladder (from top to bottom: 55 kDa, 43 kDa, 34 kDa). Elution volumes are indicated above. (<b>E</b>) Analysis of the DNM1L GMP-PNP complex stability under SEC conditions as in Fig. 8C. SEC elution (red) and further analysis of the peaks by HPLC (blue), with the indicated controls. (<b>F</b>) SEC of GG fusion protein mutants Q34A, S35A and D190A under conditions as in Fig. 8C in the presence of GDP⋅AlF₄⁻. Retention volumes of molecular weight standards are shown above.</p

Kinetic parameters of DNM1L basal GTPase activities.

a<p>In all cases except for the cooperative model with the mutant S35A, k_obs and K correspond to k_cat and K_m of the applied Michaelis-Menten model;</p>b<p>WT = 100; ^c k_obs and K could not be determined in a reliable manner, since the substrate did not reach the range of saturating levels.</p