Engineering the internal cavity of neuroglobin demonstrates the role of the haem-sliding mechanism.

Neuroglobin is a member of the globin family involved in neuroprotection; it is primarily expressed in the brain and retina of vertebrates. Neuroglobin belongs to the heterogeneous group of hexacoordinate globins that have evolved in animals, plants and bacteria, endowed with the capability of reversible intramolecular coordination, allowing the binding of small gaseous ligands (O2, NO and CO). In a unique fashion among haemoproteins, ligand-binding events in neuroglobin are dependent on the sliding of the haem itself within a preformed internal cavity, as revealed by the crystal structure of its CO-bound derivative. Point mutants of the neuroglobin internal cavity have been engineered and their functional and structural characterization shows that hindering the haem displacement leads to a decrease in CO affinity, whereas reducing the cavity volume without interfering with haem sliding has negligible functional effects.

Structural dynamics of myoglobin.

Protein structure is endowed with a complex dynamic nature, which rules function and controls activity. The experimental investigations that yield information on protein dynamics are carried out in solution; however, in most cases, the determination of protein structure is carried out by crystallography that relies on the diffraction properties of a large number of molecules, in approximately the same conformation, arranged in a three-dimensional lattice. Myoglobin, maybe the most thoroughly characterized protein, has allowed the formulation of general principles in the field of protein structure-function correlation and, since the late 1990s, it has been possible to obtain directly some insight into the complex dynamic behavior of myoglobin and other proteins by Laue diffraction. This chapter describes some of the technological features involved in obtaining reliable data by time-resolved Laue crystallography, with subnanosecond time resolution. A synopsis of the more significant findings obtained by laser photolysis of myoglobin-CO crystals is also presented, emphasizing the more general aspects of dynamics relevant to the complex energy landscape of a protein.

Neuroglobin, seven years after.

Neuroglobin is expressed in vertebrates brain and belongs to a branch of the globin family that diverged early in evolution. Sequence conservation suggests a relevant role in the nervous system, with tight structural restraints. Experiments in vivo and in vitro showed increased hypoxic stress damage upon repressing neuroglobin biosynthesis and improved recovery following overexpression. Neuroglobin shows internal heme hexacoordination, which controls oxygen affinity and kinetics. Neuroglobin concentration, oxygen affinity and enhanced autooxidation question a role in oxygen delivery; thus it was proposed that the neuroprotective effect might be due to radical scavenging or activation of protection mechanisms. Neuroglobin's structure shows a peculiar internal cavity of very large size. Binding of heme ligands is associated to a conformational change involving the heme that "slides" into the pre-existing cavity and makes the sixth coordination position available. These features may pave the way to an understanding of neuroprotection by neuroglobin.

A globin for the brain.

The discovery that a myoglobin-like hemeprotein (called neuroglobin) is expressed in our brain raised considerable curiosity from the standpoints of biochemistry and pathophysiology alike. Neuroglobin is involved in neuroprotection from damage due to hypoxia or ischemia in vitro and in vivo; overexpression of neuroglobin ameliorates the recovery from stroke in experimental animals. The mechanism underlying this remarkable effect is still mysterious. Structural studies revealed that neuroglobin has a typical globin fold, and despite being hexacoordinated, it binds reversibly O2, CO, and NO, undergoing a substantial conformational change of the heme and of the protein. The possible mechanisms involved in neuroprotection are briefly reviewed. Neuroglobin is unlikely to be involved in O2 transport (like myoglobin), although it seems to act as a sensor of the O2/NO ratio in the cell, possibly regulating the GDP/GTP exchange rate forming a specific complex with the G(alpha beta gamma)-protein when oxidized but not when bound to a gaseous ligand. Thus it appears that neuroglobin is a stress-responsive sensor for signal transduction in the brain, mediated by a ligand-linked conformational change of the protein.

The structural dynamics of myoglobin.

Conformational fluctuations in proteins were initially invoked to explain the observation that diffusion of small ligands through the matrix is a global phenomenon. Small globular proteins contain internal cavities that play a role not only in matrix dynamics but also in controlling function, tracing a pathway for the diffusion of the ligand to and from the active site. This is the main point addressed in this Review, which presents pertinent information obtained on myoglobin (Mb). Mb, a simple globular heme protein which binds reversibly oxygen and other ligands. The bond between the heme Fe(II) and gaseous ligands can be photodissociated by a laser pulse, generating a non-equilibrium population of protein structures that relaxes on a picosecond to millisecond time range. This process is associated with migration of the ligand to internal cavities of the protein, which are known to bind xenon. Some of the results obtained by laser photolysis, molecular dynamics simulations, and X-ray diffraction of intermediate states of wild-type and mutant myoglobins are summarized. The extended relaxation of the globin moiety directly observed by Laue crystallography reflects re-equilibration among conformational substates known to play an essential role in controlling protein function.

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.

Crystallization and X-ray diffraction measurements of a thermophilic archaeal recombinant amidase from Sulfolobus solfataricus MT4.

Recombinant amidase is a 55.8 kDa enzyme from the thermophilic archaeon Sulfolobus solfataricus MT4 that catalyses the hydrolysis of aliphatic amides of 2-6 C atoms as well as many aromatic amides. Single crystals of purified amidase were obtained by the hanging-drop method at 294 K. Diffraction data for the native protein (2.55 A resolution) and a putative derivative (2.20 A) have been collected at low temperature using synchrotron radiation. The crystals belong to the rhombohedral space group R3. Structure determination by multiple isomorphous replacement is in progress. It is expected that structural information from this signatured thermostable amidase will increase our knowledge of the molecular mechanisms employed to maintain high-temperature stability in thermophilic proteins.

The structures of deoxy human haemoglobin and the mutant Hb Tyralpha42His at 120 K.

The structures of deoxy human haemoglobin and an artificial mutant (Tyralpha42-->His) have been solved at 120 K. While overall agreement between these structures and others in the PDB is very good, certain side chains are found to be shifted, absent from the electron-density map or in different rotamers. Non-crystallographic symmetry (NCS) is very well obeyed in the native protein, but not around the site of the changed residue in the mutant. NCS is also not obeyed by the water molecule invariably found in the alpha-chain haem pocket in room-temperature crystal structures of haemoglobin. At 120 K, this water molecule disappears from one alpha chain in the asymmetric unit but not the other.

Crystallization and preliminary X-ray diffraction studies of a monooxygenase from Streptomyces coelicolor A3(2) involved in the biosynthesis of the polyketide actinorhodin.

The aromatic monooxygenase ActVA-Orf6 from Streptomyces coelicolor A3(2) that catalyses an unusual oxidation on the actinorhodin biosynthetic pathway has been crystallized. The crystals diffract to 1.73 A and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 46.95, b = 59.29, c = 71.67 A. Solvent-content (44%) and self-rotation function calculations predict the presence of two molecules in the asymmetric unit. Structure determination should provide further insight into the enzyme mechanism and aid in the design of biosynthetic pathways to produce new polyketide natural products with novel functionality.

The role of cavities in protein dynamics: crystal structure of a photolytic intermediate of a mutant myoglobin.

We determined the structure of the photolytic intermediate of a sperm whale myoglobin (Mb) mutant called Mb-YQR [Leu-(B10)-->Tyr; His(E7)-->Gln; Thr(E10)-->Arg] to 1.4-A resolution by ultra-low temperature (20 K) x-ray diffraction. Starting with the CO complex, illumination leads to photolysis of the Fe-CO bond, and migration of the photolyzed carbon monoxide (CO*) to a niche in the protein 8.1 A from the heme iron; this cavity corresponds to that hosting an atom of Xe when the crystal is equilibrated with xenon gas at 7 atmospheres [Tilton, R. F., Jr., Kuntz, I. D. & Petsko, G. A. (1984) Biochemistry 23, 2849-2857]. The site occupied by CO* corresponds to that predicted by molecular dynamics simulations previously carried out to account for the NO geminate rebinding of Mb-YQR observed in laser photolysis experiments at room temperature. This secondary docking site differs from the primary docking site identified by previous crystallographic studies on the photolyzed intermediate of wild-type sperm whale Mb performed at cryogenic temperatures [Teng et al. (1994) Nat. Struct. Biol. 1, 701-705] and room temperature [Srajer et al. (1996) Science 274, 1726-1729]. Our experiment shows that the pathway of a small molecule in its trajectory through a protein may be modified by site-directed mutagenesis, and that migration within the protein matrix to the active site involves a limited number of pre-existing cavities identified in the interior space of the protein.

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.

Structural dynamics of ligand diffusion in the protein matrix: A study on a new myoglobin mutant Y(B10) Q(E7) R(E10).

A triple mutant of sperm whale myoglobin (Mb) [Leu(B10) --> Tyr, His(E7) --> Gln, and Thr(E10) --> Arg, called Mb-YQR], investigated by stopped-flow, laser photolysis, crystallography, and molecular dynamics (MD) simulations, proved to be quite unusual. Rebinding of photodissociated NO, O2, and CO from within the protein (in a "geminate" mode) allows us to reach general conclusions about dynamics and cavities in proteins. The 3D structure of oxy Mb-YQR shows that bound O2 makes two H-bonds with Tyr(B10)29 and Gln(E7)64; on deoxygenation, these two residues move toward the space occupied by O2. The bimolecular rate constant for NO binding is the same as for wild-type, but those for CO and O2 binding are reduced 10-fold. While there is no geminate recombination with O2 and CO, geminate rebinding of NO displays an unusually large and very slow component, which is pretty much abolished in the presence of xenon. These results and MD simulations suggest that the ligand migrates in the protein matrix to a major "secondary site," located beneath Tyr(B10)29 and accessible via the motion of Ile(G8)107; this site is different from the "primary site" identified by others who investigated the photolyzed state of wild-type Mb by crystallography. Our hypothesis may rationalize the O2 binding properties of Mb-YQR, and more generally to propose a mechanism of control of ligand binding and dissociation in hemeproteins based on the dynamics of side chains that may (or may not) allow access to and direct temporary sequestration of the dissociated ligand in a docking site within the protein. This interpretation suggests that very fast (picosecond) fluctuations of amino acid side chains may play a crucial role in controlling O2 delivery to tissue at a rate compatible with physiology.

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.

Identification of a pattern in protein structure based on energetic and statistical considerations.

We carry out a statistical analysis of the nonbonded interactions in 10 high-resolution nonhomologous protein structures, using original algorithms. We observe a tendency of nonbonded interactions which contribute significantly (i.e., with an energy lower than the average value, referred to as "strong") to protein stability, to be concentrated in clusters of residues that are strongly sequence correlated. We characterize this sequence correlation and subsequently define a "system" as the pattern that describes these clusters. In order to study the distribution of the systems in the proteins we build a matrix for each protein and for each term of the empirical potential function used to compute the nonbonded interactions; each ij element is the number of common residues between the systems i and j. The analysis of the matrices shows the presence of compact blocks that define units in the protein structure which concentrate strong and weak interactions inside the unit itself and display relative independence with respect to the rest of the protein. Comparing the blocks defined by the three nonbonded energy components (electrostatic, hydrogen bonds, and van der Waals interactions) we observe a one-to-one correspondence between the blocks of different energy components with an average overlap of 90% of the residues forming each block.

Haemoglobin engineering. For fun and money.

The recent transplantation of an unusual allosteric effect from crocodile to human haemoglobin has implications for both molecular evolution and the engineering of artificial blood substitutes.

Engineering Ascaris hemoglobin oxygen affinity in sperm whale myoglobin: role of tyrosine B10.

The contribution to oxygen stabilization of a tyrosine residue in topological position (B10) has been studied in sperm whale myoglobin by simultaneous replacement of residues at positions (B10), (E7) and (E10) as suggested by analysis of the sequence of high oxygen affinity hemoglobins, such as that of the nematode Ascaris suum. Kinetic and equilibrium experiments with the gaseous ligands oxygen and carbon monoxide show that indeed the introduction of tyrosine (B10), together with replacement of the distal histidine (E7) with glutamine, is associated with a large decrease in the oxygen dissociation rate constant. Our results are consistent with the possible formation in the distal pocket of two hydrogen bonds with the iron-bound oxygen.

Site-directed mutagenesis in hemoglobin. Effect of some mutations at protein interfaces.

The role of selected amino acid residues in the monomer-monomer contacts of Hb A has been studied by site-directed mutagenesis of the alpha chain bearing substitutions in the subunit surfaces. Mutation alpha 38Thr-->Trp induced a stabilization of tetrameric Hb-CO with a decrease of the Kd for the equilibrium alpha 2 beta 2<==>2 alpha beta, but had not effect on ligand binding. Mutation alpha 40Thr-->Arg resulted in a complete loss of cooperativity in ligand binding. Mutation alpha 103His-->Val had no noticeable effect. We also studied the behaviour of isolated, mutated alpha chains with respect to self association: compared to wt alpha chains, mutant alpha 38Thr-->Trp showed stabilization of the dimeric state and (at high protein concentration) a detectable amount of tetramers. Mutant alpha 103His-->Val showed only a minor stabilization of the alpha 2 dimer.

Critical residues responsible for self-association differences of hemoglobin alpha and beta chains: analysis by molecular modeling.

Although the alpha and beta chains of adult human hemoglobin (Hb A) are very similar, when isolated the individual chains display marked differences in the propensities to form homotetramers: alpha chains alone associate weakly into dimers, while beta chains form relatively stable tetramers. We have examined the origin of this difference using computer-based model building and energy minimization. For oxyhemoglobin (R state) structures, interfaces have been compared for energy minimized alpha 2 beta 2, beta 4, and hypothetical alpha 4 tetramers. For the alpha 1-beta 1 interface (also designated as the X-interface) 19 alpha chain and 19 beta chain residues were identified that each contribute at least 1% to the energy of the contact in Hb A. This interface has a high degree of pseudo-symmetry, with identical residues at 6 of these positions for both chains. The geometry of the X-interface is similar for the homotetramers, with all 6 of these residues retained at the interface in beta 4 and 4 of the 6 found at the interface in alpha 4, although the alpha-alpha interface involves fewer contacts and less buried surface area. For the alpha 1-beta 2 interface (also designated as the Z-interface) 10 alpha chain and 10 beta chain residues are identified as contributing at least 1% to the energy of the contact in Hb A; about half of the contact residues are identical for corresponding positions of alpha and beta chains and most of these residues are retained at the interfaces in the two types of homotetramers, but with fewer alpha-alpha contacts.(ABSTRACT TRUNCATED AT 250 WORDS)