Structural basis of L-phosphoserine binding to Bacillus alcalophilus phosphoserine aminotransferase.

Phosphoserine aminotransferase is a vitamin B6-dependent enzyme that catalyzes the reversible conversion of 3-phosphohydroxypyruvate to L-phosphoserine using glutamate as an amine donor. In an effort to gain insight into the substrate-recognition mechanism of the enzyme, crystal structures of Bacillus alcalophilus phosphoserine aminotransferase in the presence or absence of L-phosphoserine were determined to resolutions of 1.5 and 1.6 Å, respectively. Local conformational changes induced upon substrate binding were identified. However, in contrast to other aminotransferases, no domain or subunit movements were observed. Two Arg residues (Arg42 and Arg328) and two His residues (His41 and His327) were found to form a tight binding site for the phosphate group of L-phosphoserine. Comparison with Escherichia coli phosphoserine aminotransferase in complex with the substrate analogue α-methylglutamate revealed more extensive structural changes in the case of L-phosphoserine binding. Based on the structural analysis, the flexibility of Arg328 is proposed to be critical for substrate recognition.

Conserved hydrophobic clusters on the surface of the Caf1A usher C-terminal domain are important for F1 antigen assembly.

The outer membrane usher protein Caf1A of the plague pathogen Yersinia pestis is responsible for the assembly of a major surface antigen, the F1 capsule. The F1 capsule is mainly formed by thin linear polymers of Caf1 (capsular antigen fraction 1) protein subunits. The Caf1A usher promotes polymerization of subunits and secretion of growing polymers to the cell surface. The usher monomer (811 aa, 90.5 kDa) consists of a large transmembrane ß-barrel that forms a secretion channel and three soluble domains. The periplasmic N-terminal domain binds chaperone-subunit complexes supplying new subunits for the growing fiber. The middle domain, which is structurally similar to Caf1 and other fimbrial subunits, serves as a plug that regulates the permeability of the usher. Here we describe the identification, characterization, and crystal structure of the Caf1A usher C-terminal domain (Caf1A(C)). Caf1A(C) is shown to be a periplasmic domain with a seven-stranded ß-barrel fold. Analysis of C-terminal truncation mutants of Caf1A demonstrated that the presence of Caf1A(C) is crucial for the function of the usher in vivo, but that it is not required for the initial binding of chaperone-subunit complexes to the usher. Two clusters of conserved hydrophobic residues on the surface of Caf1A(C) were found to be essential for the efficient assembly of surface polymers. These clusters are conserved between the FGL family and the FGS family of chaperone-usher systems.

Effect of pH on the structure and stability of Bacillus circulans ssp. alkalophilus phosphoserine aminotransferase: thermodynamic and crystallographic studies.

pH is one of the key parameters that affect the stability and function of proteins. We have studied the effect of pH on the pyridoxal-5'-phosphate-dependent enzyme phosphoserine aminotransferase produced by the facultative alkaliphile Bacillus circulans ssp. alkalophilus using thermodynamic and crystallographic analysis. Enzymatic activity assay showed that the enzyme has maximum activity at pH 9.0 and relative activity less than 10% at pH 7.0. Differential scanning calorimetry and circular dichroism experiments revealed variations in the stability and denaturation profiles of the enzyme at different pHs. Most importantly, release of pyridoxal-5'-phosphate and protein thermal denaturation were found to occur simultaneously at pH 6.0 in contrast to pH 8.5 where denaturation preceded cofactor's release by approximately 3 degrees C. To correlate the observed differences in thermal denaturation with structural features, the crystal structure of phosphoserine aminotransferase was determined at 1.2 and 1.5 A resolution at two different pHs (8.5 and 4.6, respectively). Analysis of the two structures revealed changes in the vicinity of the active site and in surface residues. A conformational change in a loop involved in substrate binding at the entrance of the active site has been identified upon pH change. Moreover, the number of intramolecular ion pairs was found reduced in the pH 4.6 structure. Taken together, the presented kinetics, thermal denaturation, and crystallographic data demonstrate a potential role of the active site in unfolding and suggest that subtle but structurally significant conformational rearrangements are involved in the stability and integrity of phosphoserine aminotransferase in response to pH changes.

Strain relief at the active site of phosphoserine aminotransferase induced by radiation damage.

The X-ray susceptibility of the lysine-pyridoxal-5'-phosphate Schiff base in Bacillus alcalophilus phosphoserine aminotransferase has been investigated using crystallographic data collected at 100 K to 1.3 A resolution, complemented by on-line spectroscopic studies. X-rays induce deprotonation of the internal aldimine, changes in the Schiff base conformation, displacement of the cofactor molecule, and disruption of the Schiff base linkage between pyridoxal-5'-phosphate and the Lys residue. Analysis of the "undamaged" structure reveals a significant chemical strain on the internal aldimine bond that leads to a pronounced geometrical distortion of the cofactor. However, upon crystal exposure to the X-rays, the strain and distortion are relaxed and eventually diminished when the total absorbed dose has exceeded 4.7 x 10(6) Ggamma. Our data provide new insights into the enzymatic activation of pyridoxal-5'-phosphate and suggest that special care should be taken while using macromolecular crystallography to study details in strained active sites.

Enzyme adaptation to alkaline pH: atomic resolution (1.08 A) structure of phosphoserine aminotransferase from Bacillus alcalophilus.

The crystal structure of the vitamin B(6)-dependent enzyme phosphoserine aminotransferase from the obligatory alkaliphile Bacillus alcalophilus has been determined at 1.08 A resolution. The model was refined to an R-factor of 11.7% (R(free) = 13.9%). The enzyme displays a narrow pH optimum of enzymatic activity at pH 9.0. The final structure was compared to the previously reported structure of the mesophilic phosphoserine aminotransferase from Escherichia coli and to that of phosphoserine aminotransferase from a facultative alkaliphile, Bacillus circulans subsp. alkalophilus. All three enzymes are homodimers with each monomer comprising a two-domain architecture. Despite the high structural similarity, the alkaliphilic representatives possess a set of distinctive structural features. Two residues directly interacting with pyridoxal-5'-phosphate are replaced, and an additional hydrogen bond to the O3' atom of the cofactor is present in alkaliphilic phosphoserine aminotransferases. The number of hydrogen bonds and hydrophobic interactions at the dimer interface is increased. Hydrophobic interactions between the two domains in the monomers are enhanced. Moreover, the number of negatively charged amino acid residues increases on the solvent-accessible molecular surface and fewer hydrophobic residues are exposed to the solvent. Further, the total amount of ion pairs and ion networks is significantly reduced in the Bacillus enzymes, while the total number of hydrogen bonds is increased. The mesophilic enzyme from Escherichia coli contains two additional beta-strands in a surface loop with a third beta-strand being shorter in the structure. The identified structural features are proposed to be possible factors implicated in the alkaline adaptation of phosphoserine aminotransferase.

Expression, purification, crystallization and preliminary crystallographic analysis of phosphoserine aminotransferase from Bacillus alcalophilus.

Phosphoserine aminotransferase (PSAT; EC 2.6.1.52) from Bacillus alcalophilus, an obligatory alkalophile with optimum growth at pH 10.6, was overexpressed in Escherichia coli, purified and crystallized under two different conditions using the hanging-drop vapour-diffusion method. Crystals were obtained using trisodium citrate dihydrate or PEG 400 as a precipitating agent. Crystals grown in the presence of trisodium citrate belong to the orthorhombic space group C222(1), with unit-cell parameters a = 105.6, b = 136.6, c = 152.0 A, and those grown in the presence of PEG 400 belong to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 143.7, b = 84.3, c = 67.4 A. Complete data sets were collected to 1.7 and 1.6 A resolution, respectively, at 100 K using synchrotron radiation. Analysis of the structure of B. alcalophilus PSAT may reveal structural features that contribute to enzyme adaptability at high pH values.

Antiferritin VL homodimer binds human spleen ferritin with high specificity.

The antiferritin variable light domain (VL) dimer binds human spleen ferritin ( approximately 85% L subunits) but with approximately 50-fold lower affinity, K(a)=4 x 10(7) x M(-1), than the parent F11 antibody (K(a)=2.1 x 10(9) x M(-1)). The VL dimer does not recognize either rL (100% L subunits) or rH (100% H subunits) human ferritin, whereas the parent antibody recognizes rL-ferritin. To help explain the differences in ferritin binding affinities and specificities, the crystal structure of the VL domain (2.8A resolution) was determined by molecular replacement and models of the antiferritin VL-VH dimer were made on the basis of antilysozyme antibody D1.3. The domain interface is smaller in the VL dimer but a larger number of interdomain hydrogen bonds may prevent rearrangement on antigen binding. The antigen binding surface of the VL dimer is flatter, lacking a negatively charged pocket found in the VL-VH models, contributed by the CDR3 loop of the VH domain. Loop CDR2 (VL dimer) is located away from the antigen binding site, while the corresponding loop of the VH domain would be located within the antigen binding site. Together these differences lead to 50-fold lower binding affinity in the VL dimer and to more restricted specificity than is seen for the parent antibody.

Partially structured state of the functional VH domain of the mouse anti-ferritin antibody F11.

An antibody combining site generally involves the two variable domains, VH from the heavy and VL from the light chain. We expressed the individual VH domain of the mouse anti-human ferritin monoclonal antibody F11. The loss of affinity was not dramatic (K(a)=4.0x10(7) M(-1) versus 8.6x10(8) M(-1) for the parent antibody) and comparable to that previously observed for other VHs. However, the functional VH domain adopted a partially structured state with a significant amount of distorted secondary and compact yet greatly destabilized tertiary structures, as demonstrated by spectroscopic and calorimetric probes. These data provide the first description for a functional antibody domain that meets all the criteria of a partially structured state.

Amyloid fibril formation of the mouse V(L) domain at acidic pH.

The recombinant V(L) domain that represents the variable part of the light chain (type kappa) of mouse monoclonal antibody F11 directed against human spleen ferritin was found to form amyloid fibrils at acidic pH as evidenced by electron microscopy, thioflavin T binding, and apple-green birefringence after Congo red staining. This is the first demonstration of amyloid fibril formation of the mouse V(L) domain. To understand the mechanism of acidic pH-induced amyloid fibril formation, conformational changes of the V(L) domain were studied by one-dimensional NMR, differential scanning calorimetry, analytical ultracentrifugation, hydrophobic dye binding, far-UV circular dichroism, and tryptophan fluorescence. The results indicated accumulation of two intermediate states during acid unfolding, which might be responsible for amyloid fibril formation. The more structured intermediate that exhibited maximal accumulation at pH 3 retained the nativelike secondary structure and a hydrophobic core, but exposed hydrophobic surfaces that bind 8-anilino-1-naphthalenesulfonate. Below pH 2, a more disordered intermediate with dequenched tryptophan fluorescence but still retaining the beta-sheet structure accumulated. The optimal pH of amyloid fibril formation (i.e., pH 4) was close to the optimal pH of the accumulation of the nativelike intermediate, suggesting that the amyloid fibrils might be formed through this intermediate.