Structural and functional analysis of the fibronectin-binding protein FNE from Streptococcus equi spp. equi.

Streptococcus equi is a horse pathogen belonging to Lancefield group C. Infection by S. equi ssp. equi causes strangles, a serious and highly contagious disease of the upper respiratory tract. S. equi ssp. equi secretes a fibronectin (Fn)-binding protein, FNE, that does not contain cell wall-anchoring motifs. FNE binds to the gelatin-binding domain (GBD) of Fn, composed of the motifs (6) FI (12) FII (789) FI . FNE lacks the canonical Fn-binding peptide repeats observed in many microbial surface components recognizing adhesive matrix molecules. We found that the interaction between FNE and the human GBD is mediated by the binding of the disordered C-terminal region (residues 208-262) of FNE to the (789) FI GBD subfragment. The crystal structure of FNE showed that it is similar to the minor pilus protein Spy0125 of Streptococcus pyogenes, found at the end of pilus polymers and responsible for adhesion. FNE and Spy0125 both have a superimposable internal thioester bond between highly conserved Cys and Gln residues. Small-angle X-ray scattering of the FNE-(789) FI complex provided a model that aligns the C-terminal peptide of FNE with the E-strands of the FI domains, adopting the ß-zipper extension model observed in previous structures of microbial surface components recognizing adhesive matrix molecule adhesion peptides bound to FI domains.

Redox regulation of chloroplastic G6PDH activity by thioredoxin occurs through structural changes modifying substrate accessibility and cofactor binding.

In chloroplasts, redox regulation of enzyme activities by TRXs (thioredoxins) allows the co-ordination of light/dark metabolisms such as the reductive (so-called Calvin-Benson) pathway and the OPPP (oxidative pentose phosphate pathway). Although the molecular mechanisms underlying the redox regulation of several TRX-regulated enzymes have been investigated in detail, only partial information was available for plastidial G6PDH (glucose-6-phosphate dehydrogenase) catalysing the first and rate-limiting step of the OPPP. In the present study, we investigated changes in catalytic and structural properties undergone by G6PDH1 from Arabidopsis thaliana upon treatment with TRX f1, the most efficient regulator of the enzyme that did not show a stable interaction with its target. We found that the formation of the regulatory disulfide bridge that leads to activation of the enzyme allows better substrate accessibility to the active site and strongly modifies the cofactor-binding properties. Structural modelling and data from biochemical and biophysical studies of site-directed mutant proteins support a mechanism in which the positioning/function of the highly conserved Arg(131) in the cofactor-binding site can be directly influenced by the redox state of the adjacent regulatory disulfide bridge. These findings constitute another example of modifications to catalytic properties of a chloroplastic enzyme upon redox regulation, but by a mechanism unique to G6PDH.

Affinity transfer by CDR grafting on a nonimmunoglobulin scaffold.

Neocarzinostatin (NCS) is a small "all beta" protein displaying the same overall fold as immunoglobulins. This protein possesses a well-defined hydrophobic core and two loops structurally equivalent to the CDR1 and CDR3 of immunoglobulins. NCS is the most studied member of the enediynechromoprotein family, and is clinically used as an antitumoral agent. NCS has promise as a drug delivery vehicle if new binding specificities could be conferred on its protein scaffold. Previous studies have shown that the binding specificity of the crevasse can be extended to compounds completely unrelated to the natural enediyne chromophore family. We show here that it is possible to introduce new interaction capacities to obtain a protein useful for drug targeting by modifying the immunoglobulin CDR-like loops. We transferred the CDR3 of the VHH chain of camel antilysozyme immunoglobulin to the equivalent site in the corresponding loop of neocarzinostatin. We then evaluated the stability of the resulting structure and its affinity for lysozyme. The engineered NCS-CDR3 presents a structure similar to that of the wild-type NCS, and is stable and efficiently produced. ELISA, ITC, and SPR measurements demonstrated that the new NCS-CDR3 specifically bound lysozyme.

Role of the tyrosine corner motif in the stability of neocarzinostatin.

Although the immunoglobulin-like beta-sandwich fold has no specifically conserved function, some common structural features have been observed, in particular a structural motif, the tyrosine corner. Such a motif was described in neocarzinostatin (NCS), a bacterial protein the structure of which is very similar to that of the immunoglobulin domain. Compared with the other beta-sheet proteins, the NCS 'tyrosine corner' presents non-standard structural features. To investigate the role of this motif in the NCS structure and stability, we studied the properties of a mutant where the H bond interaction had been eliminated by replacing the tyrosine with a phenylalanine. This mutation costs 4.0 kcal/mol showing that the NCS 'tyrosine corner' is involved in protein stability as in the other Greek key proteins. This destabilization is accompanied by remote structural effects, including modification of the binding properties, suggesting an increase in the internal flexibility of the protein. With a view to using this protein for drug targeting, these results along with those obtained previously allow us to define clearly the limitations of the modifications that can be performed on this scaffold.

Proton uptake of rhodobacter capsulatus reaction center mutants modified in the primary quinone environment.

Flash-induced absorbance spectroscopy was used to analyze the proton uptake and electron transfer properties of photosynthetic reaction centers (RC) of Rhodobacter capsulatus that have been genetically modified near the primary quinone electron acceptor (Q(A)). M246Ala and M247Ala, which are symmetry-related to the positions of two acidic groups, L212Glu and L213Asp, in the secondary quinone electron acceptor (QB) protein environment, have been mutated to Glu and Asp, respectively. The pH dependence of the stoichiometry of proton uptake upon formation of the P+Q(A)- (H+/P+Q(A)-) and PQ(A) (H+/Q(A)-) (P is the primary electron donor, a noncovalently linked bacteriochlorophyll dimer) states have been measured in the M246Ala --> Glu and the M247Ala --> Asp mutant RC, in the M246Ala-M247Ala --> Glu-Asp double mutant and in the wild type (WT). Our results show that the introduction of an acidic group (Glu or Asp) in the QA protein region induces notable additional proton uptake over a large pH region (approximately 6-9), which reflects a delocalized response of the protein to the formation of Q(A)-. This may indicate the existence of a widely spread proton reservoir in the cytoplasmic region of the protein. Interestingly, the pH titration curves of the proton release caused by the formation of P+ (H+/P+: difference between H+/P+Q(A)- and H+/PQ(A)- curves) are nearly superimposable in the WT and the M246Ala --> Glu mutant RC, but substantial additional proton release is detected between pH 7 and 9 in the M247Ala --> Asp mutant RC. This effect can be accounted for by an increased proton release by the P+ environment in the M247Ala --> Asp mutant. The M247Ala --> Asp mutation reveals the existence of an energetic and conformational coupling between donor and acceptor sides of the RC at a distance of nearly 30A.

Key interactions in neocarzinostatin, a protein of the immunoglobulin fold family.

Neocarzinostatin (NCS) is a seven-stranded beta-sandwich protein, the folding of which is similar to that of the variable domains of immunoglobulins (Ig). The investigation of the backbone dynamics of apo-NCS [Izadi-Pruneyre et al. (2001) Protein Sci., 10, 2228-2240] enabled us to identify the involvement of long side-chain residues in maintaining the rigidity of this beta-protein. In the perspective of using this protein for drug targeting, this raises the following question: do these residues also play a key role in the stabilization of the beta-sheet? To investigate this problem, various genetically engineered variants were constructed by mutating these residues to amino acids with shorter aliphatic side chains. These substitutions have no effects on the global fold. However, an important destabilization of the protein, higher than that expected for a simple 'large-to-small' substitution of buried hydrophobic residues, is observed for three mutants, V34A, V21A and V95A. Interestingly, the nature of the residues in these positions is highly conserved in the other Ig-like proteins. The absence of an evolutionary relationship between NCS and the other Ig-like proteins strongly suggests that this hydrophobic core is characteristic of the Ig-fold itself.