Switching between the alternative structures and functions of a 2-Cys peroxiredoxin, by site-directed mutagenesis.

Members of the typical 2-Cys peroxiredoxin (Prx) subfamily represent an intriguing example of protein moonlighting behavior since this enzyme shifts function: indeed, upon chemical stimuli, such as oxidative stress, Prx undergoes a switch from peroxidase to molecular chaperone, associated to a change in quaternary structure from dimers/decamers to higher-molecular-weight (HMW) species. In order to detail the structural mechanism of this switch at molecular level, we have designed and expressed mutants of peroxiredoxin I from Schistosoma mansoni (SmPrxI) with constitutive HMW assembly and molecular chaperone activity. By a combination of X-ray crystallography, transmission electron microscopy and functional experiments, we defined the structural events responsible for the moonlighting behavior of 2-Cys Prx and we demonstrated that acidification is coupled to local structural variations localized at the active site and a change in oligomerization to HMW forms, similar to those induced by oxidative stress. Moreover, we suggest that the binding site of the unfolded polypeptide is at least in part contributed by the hydrophobic surface exposed by the unfolding of the active site. We also find an inverse correlation between the extent of ring stacking and molecular chaperone activity that is explained assuming that the binding occurs at the extremities of the nanotube, and the longer the nanotube is, the lesser the ratio binding sites/molecular mass is.

Control of heme reactivity by diffusion: structural basis and functional characterization in hemoglobin mutants.

The effect of mutagenesis on O(2), CO, and NO binding to mutants of human hemoglobin, designed to modify some features of the reactivity that hinder use of hemoglobin solutions as blood substitute, has been extensively investigated. The kinetics may be interpreted in the framework of the Monod-Wyman-Changeux two-state allosteric model, based on the high-resolution crystallographic structures of the mutants and taking into account the control of heme reactivity by the distal side mutations. The mutations involve residues at topological position B10 and E7, i.e., Leu (B10) to Tyr and His (E7) to Gln, on either the alpha chains alone (yielding the hybrid tetramer Hbalpha(YQ)), the beta chains alone (hybrid tetramer Hbbeta(YQ)), or both types of chains (Hb(YQ)). Our data indicate that the two mutations affect ligand diffusion into the pocket, leading to proteins with low affinity for O(2) and CO, and especially with reduced reactivity toward NO, a difficult goal to achieve. The observed kinetic heterogeneity between the alpha(YQ) and beta(YQ) chains in Hb(YQ) has been rationalized on the basis of the three-dimensional structure of the active site. Furthermore, we report for the first time an experiment of partial CO binding, selective for the beta chains, to high salt crystals of the mutant Hb(YQ) in the T-state; these crystallographic data may be interpreted as "snapshots" of the initial events possibly occurring on ligand binding to the T-allosteric state of this peculiar mutant Hb.

Modulation of ligand binding in engineered human hemoglobin distal pocket.

Functional and structural studies on hemoglobin and myoglobin from different animals and engineered variants have enlightened the great importance of the physico-chemical properties of the side-chains at topological position B10 and E7. These residues proved to be crucial to the discrimination and stabilisation of gaseous ligands. In view of the data obtained on the high oxygen affinity hemoglobin from Ascaris worms and a new mutant of sperm whale myoglobin, we selected the two mutations Leu B10-->Tyr and His E7-->Gln as potentially relevant to control ligand binding parameters in the alpha and beta-chains of human hemoglobin. Here, we present an investigation of three new mutants: HbalphaYQ (alpha2YQbeta2A), HbbetaYQ (alpha2Abeta2YQ) and HbalphabetaYQ (alpha2YQbeta2YQ). They are characterised by a very low reactivity for NO, O2 and CO, and a reduced cooperativity. Their functional properties are not inconsistent with the behaviour expected for a two-state allosteric model. Proteins with these substitutions may be considered as candidates for the synthesis of a possible "blood substitute", which should yield an O2 adduct stable to autoxidation and slowly reacting with NO. The mutant HbalphabetaYQ is particularly interesting because the rate of reaction of NO with the oxy and deoxy derivatives is reduced. A structural interpretation of our data is presented based on the 3D structure of deoxy HbalphabetaYQ determined by crystallography at 1.8 A resolution.

Free energy of burying hydrophobic residues in the interface between protein subunits.

We have obtained an experimental estimate of the free energy change associated with variations at the interface between protein subunits, a subject that has raised considerable interest since the concept of accessible surface area was introduced by Lee and Richards [Lee, B. & Richards, F. M. (1971) J. Mol. Biol. 55, 379-400]. We determined by analytical ultracentrifugation the dimer-tetramer equilibrium constant of five single and three double mutants of human Hb. One mutation is at the stationary alpha1 beta1 interface, and all of the others are at the sliding alpha1 beta2 interface where cleavage of the tetramer into dimers and ligand-linked allosteric changes are known to occur. A surprisingly good linear correlation between the change in the free energy of association of the mutants and the change in buried hydrophobic surface area was obtained, after corrections for the energetic cost of losing steric complementarity at the alphabeta dimer interface. The slope yields an interface stabilization free energy of -15 +/- 1.2 cal/mol upon burial of 1 A2 of hydrophobic surface, in very good agreement with the theoretical estimate given by Eisenberg and McLachlan [Eisenberg, D. & McLachlan, A. D. (1986) Nature (London) 319, 199-203].

Probing the alpha 1 beta 2 interface of human hemoglobin by mutagenesis. Role of the FG-C contact regions.

The allosteric transition of hemoglobin involves an extensive reorganization of the alpha 1 beta 2 interface, in which two contact regions have been identified. This paper concerns at the effect of two mutations located in the "switch" (alpha C3 Thr --> Trp) and the "flexible joint" (beta C3 Trp --> Thr). We have expressed and characterized one double and two single mutants: Hb alpha T38W/beta W37T, Hb beta W37T, and Hb alpha T38W, whose structure has been determined by crystallography. We present data on: (i) the interface structure in the contact regions, (ii) oxygen and CO binding kinetics and cooperativity, (iii) dissociation rates of deoxy tetramers and association rates of deoxy dimers, and (iv) the effect of NaI on deoxy tetramer dissociation rate constant. All the mutants are tetrameric and T-state in the deoxygenated derivative. Reassociation of deoxygenated dimers is not modified by interface mutations. DeoxyHb alpha T38W/beta W37T dissociate much faster. We propose a binding site for I- at the switch region. The single mutants binds O2 cooperatively; the double one is almost non-cooperative, a feature confirmed by CO binding. The functional data, analyzed with the two-state model, indicate that these mutations reduce the value of the allosteric constant LO.