Ligand pathways in neuroglobin revealed by low-temperature photodissociation and docking experiments.

A combined biophysical approach was applied to map gas-docking sites within murine neuroglobin (Ngb), revealing snapshots of events that might govern activity and dynamics in this unique hexacoordinate globin, which is most likely to be involved in gas-sensing in the central nervous system and for which a precise mechanism of action remains to be elucidated. The application of UV-visible microspectroscopy in crystallo, solution X-ray absorption near-edge spectroscopy and X-ray diffraction experiments at 15-40 K provided the structural characterization of an Ngb photolytic intermediate by cryo-trapping and allowed direct observation of the relocation of carbon monoxide within the distal heme pocket after photodissociation. Moreover, X-ray diffraction at 100 K under a high pressure of dioxygen, a physiological ligand of Ngb, unravelled the existence of a storage site for O2 in Ngb which coincides with Xe-III, a previously described docking site for xenon or krypton. Notably, no other secondary sites were observed under our experimental conditions.

Determinants of neuroglobin plasticity highlighted by joint coarse-grained simulations and high pressure crystallography.

Investigating the effect of pressure sheds light on the dynamics and plasticity of proteins, intrinsically correlated to functional efficiency. Here we detail the structural response to pressure of neuroglobin (Ngb), a hexacoordinate globin likely to be involved in neuroprotection. In murine Ngb, reversible coordination is achieved by repositioning the heme more deeply into a large internal cavity, the "heme sliding mechanism". Combining high pressure crystallography and coarse-grain simulations on wild type Ngb as well as two mutants, one (V101F) with unaffected and another (F106W) with decreased affinity for CO, we show that Ngb hinges around a rigid mechanical nucleus of five hydrophobic residues (V68, I72, V109, L113, Y137) during its conformational transition induced by gaseous ligand, that the intrinsic flexibility of the F-G loop appears essential to drive the heme sliding mechanism, and that residue Val 101 may act as a sensor of the interaction disruption between the heme and the distal histidine.

Redirecting P450 EryK specificity by rational site-directed mutagenesis.

The C-12 hydroxylase EryK is a bacterial cytochrome P450, active during one of the final tailoring steps of erythromycin A (ErA) biosynthesis. Its tight substrate specificity, restricted to the metabolic intermediate ErD, leads to the accumulation in the culture broth of a shunt metabolite, ErB, that originates from the competitive action of a methyltranferase on the substrate of EryK. Although the methylation of the mycarosyl moiety represents the only difference between the two metabolites, EryK exhibits very low conversion of ErB in ErA via a parallel pathway. Given its limited antimicrobial activity and its moderate toxicity, contamination by such a byproduct decreases the yield and purity of the antibiotic. In this study, EryK has been redesigned to make it suitable for industrial application. Taking advantage of the three-dimensional structure of the enzyme in complex with ErD, three single active-site mutants of EryK (M86A, H88E, and E89L) have been designed to allow hydroxylation of the nonphysiological substrate ErB. The binding and catalytic properties of these three variants on both ErD and ErB have been analyzed. Interestingly, we found the mutation of Met 86 to Ala to yield enzymatic activity on both ErB and ErD. The three-dimensional structure of the complex of mutated EryK with ErB revealed that the mutation allows ErB to accommodate in the active site of the enzyme and to induce its closure, thus assuring the progress of the catalytic reaction. Therefore, by single mutation the fine substrate recognition, active site closure, and locking were recovered.

Binding properties of the calcium-activated F2 isoform of Lethocerus troponin C.

While in most muscles contraction is triggered by calcium effluxes, insect flight muscles are also activated by mechanical stretch. We are interested in understanding the role that the troponin C protein, usually the calcium sensor, plays in stretch activation. In the flight muscles of Lethocerus, a giant water bug often used as a model system, there are two isoforms of TnC, F1 and F2, present in an approximately 10:1 ratio. F1 TnC is responsible for activating the muscle following a stretch, whereas F2 TnC produces a sustained contraction, the magnitude of which depends on the concentration of Ca(2+) in the fiber. We have previously shown that F1 TnC binds only one Ca(2+) ion in its C-terminal domain and that interaction with troponin H, the insect ortholog of troponin I, is insensitive to Ca(2+). Here, we have studied the effect of Ca(2+) and Mg(2+) on the affinities of the interaction of F2 TnC with troponin H peptides. We show that the presence of two Ca(2+) ions, one in each of the globular domains, increases the affinity for TnH by at least 1 order of magnitude. The N lobe has a lower affinity for Ca(2+), but it is also sensitive to Mg(2+). The C lobe is insensitive to Mg(2+) as previously demonstrated by mutations of the individual EF-hands. The interaction with TnH seems also to have significant structural differences from that observed for the F1 TnC isoform. We discuss how our findings could account for stretch activation.

Structural features for the mechanism of antitumor action of a dimeric human pancreatic ribonuclease variant.

A specialized class of RNases shows a high cytotoxicity toward tumor cell lines, which is critically dependent on their ability to reach the cytosol and to evade the action of the ribonuclease inhibitor (RI). The cytotoxicity and antitumor activity of bovine seminal ribonuclease (BSRNase), which exists in the native state as an equilibrium mixture of a swapped and an unswapped dimer, are peculiar properties of the swapped form. A dimeric variant (HHP2-RNase) of human pancreatic RNase, in which the enzyme has been engineered to reproduce the sequence of BSRNase helix-II (Gln28-->Leu, Arg31-->Cys, Arg32-->Cys, and Asn34-->Lys) and to eliminate a negative charge on the surface (Glu111-->Gly), is also extremely cytotoxic. Surprisingly, this activity is associated also to the unswapped form of the protein. The crystal structure reveals that on this molecule the hinge regions, which are highly disordered in the unswapped form of BSRNase, adopt a very well-defined conformation in both subunits. The results suggest that the two hinge peptides and the two Leu28 side chains may provide an anchorage to a transient noncovalent dimer, which maintains Cys31 and Cys32 of the two subunits in proximity, thus stabilizing a quaternary structure, similar to that found for the noncovalent swapped dimer of BSRNase, that allows the molecule to escape RI and/or to enhance the formation of the interchain disulfides.