Structural Studies on Bacteriophage Portal Proteins

In tailed bacteriophages and evolutionarily related herpes viruses, the portal protein is a central component of the DNA packaging molecular motor, which translocates viral genomic DNA into a preformed procapsid. The motor is the most powerful molecular machine discovered in nature, generating forces reaching ~50 pN and translocating DNA with a speed of several hundred bp/sec using ATP as an energy source. The oligomeric portal protein ring is situated at a unique vertex of the procapsid forming a conduit for DNA entry and exit. Although the three-dimensional structure has already been determined for portal proteins from bacteriophages P22, SPP1 and phi29, several important questions about the role of individual protein segments in DNA translocation and their interaction with other components of the motor remain unanswered. Structural information on portal proteins from other bacteriophages, like T4 for which a wealth of biochemical information is already available, will help to answer at least some of these questions. The portal protein of bacteriophage SPP1 (gp6) can form circular oligomers containing 12 or 13 subunits. It is found as a 12-subunit oligomer when incorporated into the viral capsid and as a 13-subunit assembly in its isolated form. The X-ray structure of the SPP1 portal protein is available only for the isolated 13-subunit assembly of the N365K mutant form. Because this mutation results in a reduction in the length of packaged DNA, determining the structure of the wild type portal protein would shed light on the mechanism of DNA translocation. Elucidation of the mechanism of DNA packaging depends also on the availability of accurate structural information on the SPP1 portal protein in its 12-subunit biologically active state. Such structural knowledge would be particularly useful in future, for designing a stable molecular machine that can function in vitro. In this thesis, experiments were designed to promote the formation of the dodecameric gp6: viz fusing gp6 with TRAP protein that forms a stable circular dodecamer as well as the co-expression of gp6 with the SPP1 scaffolding protein gp11. The protein targets were cloned, expressed and purified, and the oligomeric state of gp6 was characterised by a combination of biochemical, biophysical and structural approaches. The structure of the wild type gp6 was solved at 2.8 Å resolution, revealing a 13-fold symmetrical molecule. The protein’s fold is the same as for the N365K mutant, with most significant conformational differences observed in the tunnel loop and in segments of the clip and crown domains. Comparison with the structure of N365K mutant reveals significant differences in subunit-subunit interactions formed by tunnel loops, including different hydrogen bonding and van der Waals interactions. It is likely that these differences account for the different amount of packaged DNA, indicating involvement of tunnel loops in DNA packaging. The portal protein of bacteriophage CNPH82, cn3, was also successfully cloned, expressed and purified. Promising crystallisation conditions have been identified that yield crystals diffracting to 4.2 Å. Further optimisation should lead to determination of the X-ray structure of this protein in not too distant future. Self-rotation function calculations and SEC-MALLS analysis indicate that the cn3 protein forms 13-subunit assemblies, in common with the SPP1 portal protein. Foundation work has been carried out for the bacteriophage T4 portal protein, aimed at identifying suitable production and purification conditions. In addition, the full-length bacteriophage SPP1 scaffolding protein gp11 has been cloned, purified and crystallised. Degradation was observed in the full length gp11 protein and therefore a series of truncations were designed, cloned and purified aiming to improve the stability. Further studies on limited proteolysis of the full-length gp11 should lead to a stable gp11 tuncation that will form crystals with better diffraction

Structure and substrate specificity determinants of the taurine biosynthetic enzyme cysteine sulphinic acid decarboxylase

Abstract Pyridoxal 5́-phosphate (PLP) is an important cofactor for amino acid decarboxylases with many biological functions, including the synthesis of signalling molecules, such as serotonin, dopamine, histamine, γ-aminobutyric acid, and taurine. Taurine is an abundant amino acid with multiple physiological functions, including osmoregulation, pH regulation, antioxidative protection, and neuromodulation. In mammalian tissues, taurine is mainly produced by decarboxylation of cysteine sulphinic acid to hypotaurine, catalysed by the PLP-dependent cysteine sulphinic acid decarboxylase (CSAD), followed by oxidation of the product to taurine. We determined the crystal structure of mouse CSAD and compared it to other PLP-dependent decarboxylases in order to identify determinants of substrate specificity and catalytic activity. Recognition of the substrate involves distinct side chains forming the substrate-binding cavity. In addition, the backbone conformation of a buried active-site loop appears to be a critical determinant for substrate side chain binding in PLP-dependent decarboxylases. Phe94 was predicted to affect substrate specificity, and its mutation to serine altered both the catalytic properties of CSAD and its stability. Using small-angle X-ray scattering, we further showed that CSAD presents open/close motions in solution. The structure of apo-CSAD indicates that the active site gets more ordered upon internal aldimine formation. Taken together, the results highlight details of substrate recognition in PLP-dependent decarboxylases and provide starting points for structure-based inhibitor design with the aim of affecting the biosynthesis of taurine and other abundant amino acid metabolites

Structure of the mouse acidic amino acid decarboxylase GADL1

Abstract Pyridoxal 5′-phosphate (PLP) is a ubiquitous cofactor in various enzyme classes, including PLP-dependent decarboxylases. A recently discovered member of this class is glutamic acid decarboxylase-like protein 1 (GADL1), which lacks the activity to decarboxylate glutamate to γ-aminobutyrate, despite its homology to glutamic acid decarboxylase. Among the acidic amino acid decarboxylases, GADL1 is most similar to cysteine sulfinic acid decarboxylase (CSAD), but the physiological function of GADL1 is unclear, although its expression pattern and activity suggest a role in neurotransmitter and neuroprotectant metabolism. The crystal structure of mouse GADL1 is described, together with a solution model based on small-angle X-ray scattering data. While the overall fold and the conformation of the bound PLP are similar to those in other PLP-dependent decarboxylases, GADL1 adopts a more loose conformation in solution, which might have functional relevance in ligand binding and catalysis. The structural data raise new questions about the compactness, flexibility and conformational dynamics of PLP-dependent decarboxylases, including GADL1

Data collection and refinement statistics for <i>B. subtilis</i> S72N TRAP.

<p>Values in parentheses are for the highest resolution shell.</p>a<p><i>R_merge</i>  =  ∑<i>_hkl</i>∑_i|<i>I_i(h)</i> - <<i>I(h)></i>|/∑<i>_hkl</i>∑_i<i>I_i(h)</i>, where <i>I(h)</i> is intensity of reflection <i>h</i>, <<i>I(h)></i> is average value of intensity, the sum ∑<i>_hkl</i> is over all measured reflections and the sum ∑_i is over <i>i</i> measurements of a reflection.</p>b<p>Crystallographic <i>R  = </i> ∑<i>_hkl</i>||<i>F_obs</i> - <i>F_calc</i>||/∑<i>_hkl</i>|<i>F_obs|</i>, <i>R_free</i> was calculated using a randomly chosen set of reflections that were excluded from the refinement.</p

Melting temperatures of different TRAP oligomers.

<p>The dependence of melting temperature is shown for six TRAP oligomers, as a function of L-tryptophan concentration, determined by dye-based scanning fluorimetry. From the top: <i>B. stearothermophilus</i> E71stop 12-mer (triangle), <i>B. stearothermophilus</i> wild type 11-mer (square), <i>B. halodurans</i> wild type 12-mer (diamond), <i>B. subtilis</i> K71stop 12-mer (open cross), <i>B. subtilis</i> S72N 12-mer (circle), <i>B. subtilis</i> wild type 11-mer (crossed square).</p

Structural differences between 11-subunit and 12-subunit TRAP.

<p>(<b>A</b>) Comparison of the <i>B. subtilis</i> wild type (red) and S72N mutant TRAP (blue). Two neighboring subunits were least-square fitted using main chain atoms of the subunit on the left. The C-terminal residues that are pivoted out of the subunit interface in S72N TRAP are highlighted in white on the wild type TRAP, starting from residue 69. (<b>B</b>) C-terminal residues E69 and M70 are shown in sticks with main chain in yellow and side chains in turquoise, the rest of each subunit is shown in ribbons. The weighted <i>F</i>_o – <i>F</i>_c omit maps, contoured at 2σ, were calculated after omitting residues 69 and 70 from the final model and 10 cycles of refinement.</p

Ribbon diagram of the <i>B. subtilis</i> S72N TRAP viewed along the 12-fold axis.

<p>Each subunit is shown in a different colour. L-tryptophan molecules are shown as van der Waals models with oxygen atoms in red, nitrogen atoms in blue and carbon atoms in yellow.</p

L-tryptophan binding constants (K_d) of three wild type and three mutant TRAP.

<p>L-tryptophan binding constants (K_d) of three wild type and three mutant TRAP.</p