The leghemoglobin proximal heme pocket directs oxygen dissociation and stabilizes bound heme

Sperm whale myoglobin (Mb) and soybean leghemoglobin (Lba) are two small, monomeric hemoglobins that share a common globin fold but differ widely in many other aspects. Lba has a much higher affinity for most ligands, and the two proteins use different distal and proximal heme pocket regulatory mechanisms to control ligand binding. Removal of the constraint provided by covalent attachment of the proximal histidine to the F-helices of these proteins decreases oxygen affinity in Lba and increases oxygen affinity in Mb, mainly because of changes in oxygen dissociation rate constants. Hence, Mb and Lba use covalent constraints in opposite ways to regulate ligand binding. Swapping the F-helices of the two proteins brings about similar effects, highlighting the importance of this helix in proximal heme pocket regulation of ligand binding. The F7 residue in Mb is capable of weaving a hydrogen-bonding network that holds the proximal histidine in a fixed orientation. On the contrary, the F7 residue in Lba lacks this property and allows the proximal histidine to assume a conformation favorable for higher ligand binding affinity. Geminate recombination studies indicate that heme iron reactivity on picosecond timescales is not the dominant cause for the effects observed in each mutation. Results also indicate that in Lba the proximal and distal pocket mutations probably influence ligand binding independently. These results are discussed in the context of current hypotheses for proximal heme pocket structure and function.     

Open and closed conformation of the E. coli purine nucleoside phosphorylase active center and implications for the catalytic mechanism.

The crystal structure of the ternary complex of hexameric purine nucleoside phosphorylase (PNP) from Escherichia coli with formycin A derivatives and phosphate or sulphate ions is determined at 2.0 A resolution. The hexamer is found as a trimer of unsymmetric dimers, which are formed by pairs of monomers with active sites in different conformations. The conformational difference stems from a flexible helix (H8: 214-236), which is continuous in one conformer, and segmented in the other. With the continuous helix, the entry into the active site pocket is wide open, and the ligands are bound only loosely ("open" or "loose binding" conformation). By segmentation of the helix (H8: 214-219 and H8': 223-236, separated by a gamma-turn), the entry into the active site is partially closed, the pocket is narrowed and the ligands are bound much more tightly ("closed" or "tight binding" conformation). Furthermore, the side-chain of Arg217 is carried by the moving helix into the active site. This residue, conserved in all homologous PNPs, plays an important role in the proposed catalytic mechanism. In this mechanism, substrate binding takes place in the open, and and the catalytic action occurs in the closed conformation. Catalytic action involves protonation of the purine base at position N7 by the side-chain of Asp204, which is initially in the acid form. The proton transfer is triggered by the Arg217 side-chain which is moved by the conformation change into hydrogen bond distance to Asp204. The mechanism explains the broad specificity of E. coli PNP, which allows 6-amino as well as 6-oxo-nucleosides as substrates. The observation of two kinds of binding sites is fully in line with solution experiments which independently observe strong and weak binding sites for phosphate as well as for the nucleoside inhibitor.     

Structures of the alpha L I domain and its complex with ICAM-1 reveal a shape-shifting pathway for integrin regulation

The structure of the I domain of integrin alpha L beta 2 bound to the Ig superfamily ligand ICAM-1 reveals the open ligand binding conformation and the first example of an integrin-IgSF interface. The I domain Mg2+ directly coordinates Glu-34 of ICAM-1, and a dramatic swing of I domain residue Glu-241 enables a critical salt bridge. Liganded and unliganded structures for both high- and intermediate-affinity mutant I domains reveal that ligand binding can induce conformational change in the alpha L I domain and that allosteric signals can convert the closed conformation to intermediate or open conformations without ligand binding. Pulling down on the C-terminal alpha 7 helix with introduced disulfide bonds ratchets the beta 6-alpha 7 loop into three different positions in the closed, intermediate, and open conformations, with a progressive increase in affinity.     

Molecular mechanism for ligand stabilization in the mollusc myoglobin possessing the distal Val residue.

Myoglobin extracted from the triturative stomach of Dolabella auricularia, a common mollusc found on the Japanese coast, possesses naturally occurring substitution at the distal E7 position (Val-E7) and its oxygen affinity is only slightly lower than those of the common mammalian myoglobins possessing the usual His-E7. 1H nuclear magnetic resonance studies of Dolabella met-cyano myoglobin have revealed that a guanidino NH proton of Arg-E10 is hydrogen-bonded to the Fe-bound CN-. The role of Arg-E10 as a hydrogen-bond donor for Fe-bound ligand in the present myoglobin appears to be responsible for its relatively high ligand affinity.     

Crystal structures of myoglobin-ligand complexes at near-atomic resolution.

We have used x-ray crystallography to determine the structures of sperm whale myoglobin (Mb) in four different ligation states (unligated, ferric aquomet, oxygenated, and carbonmonoxygenated) to a resolution of better than 1.2 A. Data collection and analysis were performed in as much the same way as possible to reduce model bias in differences between structures. The structural differences among the ligation states are much smaller than previously estimated, with differences of <0.25 A root-mean-square deviation among all atoms. One structural parameter previously thought to vary among the ligation states, the proximal histidine (His-93) azimuthal angle, is nearly identical in all the ferrous complexes, although the tilt of the proximal histidine is different in the unligated form. There are significant differences, however, in the heme geometry, in the position of the heme in the pocket, and in the distal histidine (His-64) conformations. In the CO complex the majority conformation of ligand is at an angle of 18 +/- 3 degrees with respect to the heme plane, with a geometry similar to that seen in encumbered model compounds; this angle is significantly smaller than reported previously by crystallographic studies on monoclinic Mb crystals, but still significantly larger than observed by photoselection. The distal histidine in unligated Mb and in the dioxygenated complex is best described as having two conformations. Two similar conformations are observed in MbCO, in addition to another conformation that has been seen previously in low-pH structures where His-64 is doubly protonated. We suggest that these conformations of the distal histidine correspond to the different conformational substates of MbCO and MbO(2) seen in vibrational spectra. Full-matrix refinement provides uncertainty estimates of important structural parameters. Anisotropic refinement yields information about correlated disorder of atoms; we find that the proximal (F) helix and heme move approximately as rigid bodies, but that the distal (E) helix does not.     

Extracellular protons titrate voltage gating of a ligand-gated ion channel

Cyclic nucleotide–gated channels mediate transduction of light into electric signals in vertebrate photoreceptors. These channels are primarily controlled by the binding of intracellular cyclic GMP (cGMP). Glutamate residue 363 near the extracellular end of the ion selectivity filter interacts with the pore helix and helps anchor the filter to the helix. Disruption of this interaction by mutations renders the channels essentially fully voltage gated in the presence of saturating concentrations of cGMP. Here, we find that lowering extracellular pH makes the channels conduct in an extremely outwardly rectifying manner, as does a neutral glutamine substitution at E363. A pair of cysteine mutations, E363C and L356C (the latter located midway the pore helix), largely eliminates current rectification at low pH. Therefore, this low pH-induced rectification primarily reflects voltage-dependent gating involving the ion selectivity filter rather than altered electrostatics around the external opening of the ion pore and thus ion conduction. It then follows that protonation of E363, like the E363Q mutation, disrupts the attachment of the selectivity filter to the pore helix. Loosening the selectivity filter from its surrounding structure shifts the gating equilibrium toward closed states. At low extracellular pH, significant channel opening occurs only when positive voltages drive the pore from a low probability open conformation to a second open conformation. Consequently, at low extracellular pH the channels become practically fully voltage gated, even in the presence of a saturating concentration of cGMP.     

Structure of unliganded GRP94, the endoplasmic reticulum Hsp90. Basis for nucleotide-induced conformational change

GRP94, the endoplasmic reticulum paralog of Hsp90, is regulated by adenosine nucleotides that bind to its N-terminal regulatory domain. Because of its weak affinity for nucleotides, the functionally relevant transition in GRP94 is likely to be between the unliganded and nucleotide-bound states. We have determined the structure of the unliganded GRP94 N-domain. The helix 1-4-5 subdomain of the unliganded protein adopts the closed conformation seen in the structure of the protein in complex with inhibitors. This conformation is distinct from the open conformation of the subdomain seen when the protein is bound to ATP or ADP. ADP soaked into crystals of the unliganded protein reveals an intermediate conformation midway between the open and closed states and demonstrates that in GRP94 the conversion between the open and closed states is driven by ligand binding. The direction of the observed movement in GRP94 shows that nucleotides act to open the subdomain elements rather than close them, which is contrary to the motion proposed for Hsp90. These observations support a model where ATP binding dictates the conformation of the N-domain and regulates its ability to form quaternary structural interactions.     

Effect of the distal residues on the vibrational modes of the Fe-CO bond in hemoglobin studied by protein engineering

Using an Escherichia coli gene expression system, we have engineered human hemoglobin (Hb) mutants having the distal histidine (E7) and valine (E11) residues replaced by other amino acids. The interaction between the mutated distal residues and bound carbon monoxide has been studied by Soret-excited resonance Raman spectroscopy. The replacement of Val-E11 by Ala, Leu, Ile, and Met has no effect on the v(C-O), v(Fe-CO) stretching or delta(Fe-C-O) bending frequencies in both the alpha and beta subunits of Hb, although some of these mutations affect the CO affinity as much as 40-fold. The strain imposed on the protein by the binding of CO is not localized in the Fe-CO bond and is probably distributed among many bonds in the globin. The replacement of His-E7 by Val or Gly brings the stretching frequencies v(Fe-CO) and v(C-O) close to those of free heme complexes. In contrast, the substitution of His-E7 by Gln, which is flexible and polar, produces no effects on the resonance Raman spectrum of either alpha- or beta-globin. The replacement of His-E7 of beta-globin by Phe shows the same effect as replacement by Gly or Val. Therefore, the steric bulk of the distal residues is not the primary determinant of the Fe-CO ligand vibrational frequencies. The ability of both histidine and glutamine to alter the v(C-O), v(Fe-CO), or delta(Fe-C-O) frequencies may be attributed to the polar nature of their side chains which can interact with bound CO in a similar manner.     

Proximal ligand control of heme iron coordination structure and reactivity with hydrogen peroxide: investigations of the myoglobin cavity mutant H93G with unnatural oxygen donor proximal ligands

The role of the proximal heme iron ligand in activation of hydrogen peroxide and control of spin state and coordination number in heme proteins is not yet well understood. Although there are several examples of amino acid sidechains with oxygen atoms which can act as potential heme iron ligands, the occurrence of protein-derived oxygen donor ligation in natural protein systems is quite rare. The sperm whale myoglobin cavity mutant H93G Mb (D. Barrick, Biochemistry 33 (1994) 6546) has its proximal histidine ligand replaced by glycine, a mutation which leaves an open cavity capable of accommodation of a variety of unnatural potential proximal ligands. This provides a convenient system for studying ligand-protein interactions. Molecular modeling of the proximal cavity in the active site of H93G Mb indicates that the cavity is of sufficient size to accommodate benzoate and phenolate in conformations that allow their oxygen atoms to come within binding distance of the heme iron. In addition, benzoate may occupy the cavity in an orientation which allows one carboxylate oxygen atom to ligate to the heme iron while the other carboxylate oxygen is within hydrogen bonding distance of serine 92. The ferric phenolate and benzoate complexes have been prepared and characterized by UV-visible and MCD spectroscopies. The benzoate adduct shows characteristics of a six-coordinate high-spin complex. To our knowledge, this is the first known example of a six-coordinate high-spin heme complex with an anionic oxygen donor proximal ligand. The benzoate ligand is displaced at alkaline pH and upon reaction with hydrogen peroxide. The phenolate adduct of H93G Mb is a five-coordinate high-spin complex whose UV-visible and MCD spectra are distinct from those of the histidine 93 to tyrosine (H93Y Mb) mutant of sperm whale myoglobin. The phenolate adduct is stable at alkaline pH and exhibits a reduced reactivity with hydrogen peroxide relative to that of both native ferric myoglobin, and the exogenous ligand-free derivative of ferric H93G Mb. These observations indicate that the identity of the proximal oxygen donor ligand has an important influence on both the heme iron coordination number and the reactivity of the complex with hydrogen peroxide.     

Energetics of the cleft closing transition and the role of electrostatic interactions in conformational rearrangements of the glutamate receptor ligand binding domain

The ionotropic glutamate receptors are localized in the pre- and postsynaptic membrane of neurons in the brain. Activation by the principal excitatory neurotransmitter glutamate allows the ligand binding domain to change conformation, communicating opening of the channel for ion conduction. The free energy of the GluR2 S1S2 ligand binding domain (S1S2) closure transition was computed using a combination of thermodynamic integration and umbrella sampling modeling methods. A path that involves lowering the charge on E705 was chosen to clarify the role of this binding site residue. A continuum electrostatics approach in S1S2 is used to show E705, located in the ligand binding cleft, stabilizes the closed conformation of S1S2 via direct interactions with other protein residues, not through the ligand. In the closed conformation, in the absence of a ligand, S1S2 is somewhat more closed than what has been reported in X-ray structures. A semiopen conformation has been identified which is characterized by disruption of a single cross-cleft interaction and differs only slightly in energy from the fully closed S1S2. The fully open S1S2 conformation exhibits a wide energy well and shares structural similarity with the apo S1S2 crystal structure. Hybrid continuum electrostatics/MD calculations along the chosen closure transition pathway reveal solvation energies, and electrostatic interaction energies between two lobes of the protein increase the relative energetic difference between the open and closed conformational states. By analyzing the role of several cross-cleft contacts as well as other binding site residues, we demonstrate how S1S2 interactions facilitate formation of the closed conformation of the GluR2 ligand binding domain.     

Induced fit movements and metal cofactor selectivity of class II aldolases: structure of Thermus aquaticus fructose-1,6-bisphosphate aldolase

Fructose-1,6-bisphosphate (FBP) aldolase is an essential glycolytic enzyme that reversibly cleaves its ketohexose substrate into triose phosphates. Here we report the crystal structure of a metallo-dependent or class II FBP aldolase from an extreme thermophile, Thermus aquaticus (Taq). The quaternary structure reveals a tetramer composed of two dimers related by a 2-fold axis. Taq FBP aldolase subunits exhibit two distinct conformational states corresponding to loop regions that are in either open or closed position with respect to the active site. Loop closure remodels the disposition of chelating active site histidine residues. In subunits corresponding to the open conformation, the metal cofactor, Co(2+), is sequestered in the active site, whereas for subunits in the closed conformation, the metal cation exchanges between two mutually exclusive binding loci, corresponding to a site at the active site surface and an interior site vicinal to the metal-binding site in the open conformation. Cofactor site exchange is mediated by rotations of the chelating histidine side chains that are coupled to the prior conformational change of loop closure. Sulfate anions are consistent with the location of the phosphate-binding sites of the FBP substrate and determine not only the previously unknown second phosphate-binding site but also provide a mechanism that regulates loop closure during catalysis. Modeling of FBP substrate into the active site is consistent with binding by the acyclic keto form, a minor solution species, and with the metal cofactor mediating keto bond polarization. The Taq FBP aldolase structure suggests a structural basis for different metal cofactor specificity than in Escherichia coli FBP aldolase structures, and we discuss its potential role during catalysis. Comparison with the E. coli structure also indicates a structural basis for thermostability by Taq FBP aldolase.     

Proton coupling and the multiscale kinetic mechanism of a peptide transporter

Proton-coupled peptide transporters (POTs) are crucial for the uptake of di- and tripeptides as well as drug and prodrug molecules in prokaryotes and eukaryotic cells. We illustrate from multiscale modeling how transmembrane proton flux couples within a POT protein to drive essential steps of the full functional cycle: 1) protonation of a glutamate on transmembrane helix 7 (TM7) opens the extracellular gate, allowing ligand entry; 2) inward proton flow induces the cytosolic release of ligand by varying the protonation state of a second conserved glutamate on TM10; 3) proton movement between TM7 and TM10 is thermodynamically driven and kinetically permissible via water proton shuttling without the participation of ligand. Our results, for the first time, give direct computational confirmation for the alternating access model of POTs, and point to a quantitative multiscale kinetic picture of the functioning protein mechanism.     

Flavohemoglobin, a globin with a peroxidase-like catalytic site

Biochemical studies of flavohemoglobin (Hmp) from Escherichia coli suggest that instead of aerobic oxygen delivery, a dioxygenase converts NO to NO3(-) and anaerobically, an NO reductase converts NO to N(2)O. To investigate the structural features underlying the chemical reactivity of Hmp, we have measured the resonance Raman spectra of the ligand-free ferric and ferrous protein and the CO derivatives of the ferrous protein. At neutral pH, the ferric protein has a five-coordinate high-spin heme, similar to peroxidases. In the ferrous protein, a strong iron-histidine stretching mode is present at 244 cm(-1). This frequency is much higher than that of any other globin discovered to date, although it is comparable to those of peroxidases, suggesting that the proximal histidine has imidazolate character. In the CO derivative, an open and a closed conformation were detected. The distal environment of the closed conformation is very polar, where the heme-bound CO strongly interacts with the B10 Tyr and/or the E7 Gln. These data demonstrate that the active site structure of Hmp is very similar to that of peroxidases and is tailored to perform oxygen chemistry.     

Alteration of sperm whale myoglobin heme axial ligation by site-directed mutagenesis

Three mutant proteins of sperm whale myoglobin (Mb) that exhibit altered axial ligations were constructed by site-directed mutagenesis of a synthetic gene for sperm whale myoglobin. Substitution of distal pocket residues, histidine E7 and valine E11, with tyrosine and glutamic acid generated His(E7)Tyr Mb and Val(E11)Glu Mb. The normal axial ligand residue, histidine F8, was also replaced with tyrosine, resulting in His(F8)Tyr Mb. These proteins are analogous in their substitutions to the naturally occurring hemoglobin M mutants (HbM). Tyrosine coordination to the ferric heme iron of His(E7)Tyr Mb and His(F8)Tyr Mb is suggested by optical absorption and EPR spectra and is verified by similarities to resonance Raman spectral bands assigned for iron-tyrosine proteins. His(E7)Tyr Mb is high-spin, six-coordinate with the ferric heme iron coordinated to the distal tyrosine and the proximal histidine, resembling Hb M Saskatoon [His(beta E7)Tyr], while the ferrous iron of this Mb mutant is high-spin, five-coordinate with ligation provided by the proximal histidine. His(F8)Tyr Mb is high-spin, five-coordinate in both the oxidized and reduced states, with the ferric heme iron liganded to the proximal tyrosine, resembling Hb M Iwate [His(alpha F8)Tyr] and Hb M Hyde Park [His(beta F8)Tyr]. Val(E11)Glu Mb is high-spin, six-coordinate with the ferric heme iron liganded to the F8 histidine. Glutamate coordination to the ferric iron of this mutant is strongly suggested by the optical and EPR spectral features, which are consistent with those observed for Hb M Milwaukee [Val(beta E11)Glu]. The ferrous iron of Val(E11)Glu Mb exhibits a five-coordinate structure with the F8 histidine-iron bond intact.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure of complement component C2A: implications for convertase formation and substrate binding

C2a provides the catalytic center to the convertase complexes of the classical and lectin-binding pathways of complement activation. We determined two crystal structures of full-length C2a, with and without a pseudo ligand bound. Both structures reveal a near-active conformation of the catalytic center of the serine protease domains, while the von Willebrand factor A-type domains display an intermediate activation state of helix alpha7 with an open, activated metal-ion-dependent adhesion site. The open adhesion site likely serves to enhance the affinity for the ligand C4b, similar to "inside-out" signaling in integrins. Surprisingly, the N-terminal residues of C2a are buried in a crevice near helix alpha7, indicative of a structural switch between C2 and C2a. Extended loops on the protease domain possibly envelop the protruding anaphylatoxin domain of the substrate C3. Together with a putative substrate-induced completion of the oxyanion hole, this may contribute to the high substrate specificity of the convertases.     

Structural studies of the streptavidin binding loop.

The streptavidin-biotin complex provides the basis for many important biotechnological applications and is an interesting model system for studying high-affinity protein-ligand interactions. We report here crystallographic studies elucidating the conformation of the flexible binding loop of streptavidin (residues 45 to 52) in the unbound and bound forms. The crystal structures of unbound streptavidin have been determined in two monoclinic crystal forms. The binding loop generally adopts an open conformation in the unbound species. In one subunit of one crystal form, the flexible loop adopts the closed conformation and an analysis of packing interactions suggests that protein-protein contacts stabilize the closed loop conformation. In the other crystal form all loops adopt an open conformation. Co-crystallization of streptavidin and biotin resulted in two additional, different crystal forms, with ligand bound in all four binding sites of the first crystal form and biotin bound in only two subunits in a second. The major change associated with binding of biotin is the closure of the surface loop incorporating residues 45 to 52. Residues 49 to 52 display a 3(10) helical conformation in unbound subunits of our structures as opposed to the disordered loops observed in other structure determinations of streptavidin. In addition, the open conformation is stabilized by a beta-sheet hydrogen bond between residues 45 and 52, which cannot occur in the closed conformation. The 3(10) helix is observed in nearly all unbound subunits of both the co-crystallized and ligand-free structures. An analysis of the temperature factors of the binding loop regions suggests that the mobility of the closed loops in the complexed structures is lower than in the open loops of the ligand-free structures. The two biotin bound subunits in the tetramer found in the MONO-b1 crystal form are those that contribute Trp 120 across their respective binding pockets, suggesting a structural link between these binding sites in the tetramer. However, there are no obvious signatures of binding site communication observed upon ligand binding, such as quaternary structure changes or shifts in the region of Trp 120. These studies demonstrate that while crystallographic packing interactions can stabilize both the open and closed forms of the flexible loop, in their absence the loop is open in the unbound state and closed in the presence of biotin. If present in solution, the helical structure in the open loop conformation could moderate the entropic penalty associated with biotin binding by contributing an order-to-disorder component to the loop closure.     

Investigating the accessibility of the closed domain conformation of citrate synthase using essential dynamics sampling

A molecular dynamics study of pig heart citrate synthase is presented that aims to directly address the question of whether, for this enzyme, the ligand-induced closed domain conformation is accessible to the open unliganded enzyme. The approach utilises the technique of essential dynamics sampling, which is used in two modes. In exploring mode, the enzyme is encouraged to explore domain conformations it might not normally sample in free molecular dynamics simulation. In targeting mode, the enzyme is encouraged to adopt the domain conformation of a target structure. Using both modes extensively, it has been found that when the enzyme is prepared from a crystallographic open-domain structure and is in the unliganded state, it is unable to adopt the crystallographic closed-domain conformation of the liganded enzyme. Likewise, when the enzyme is prepared from the crystallographic closed liganded conformation with the ligands removed, it is unable to adopt the crystallographic open domain conformation. Structural investigations point to a common structural difference that is the source of this energy barrier; namely, the shift of alpha-helix 328-341 along its own axis relative to the large domain. Without this shift, the domains are unable to close or open fully. The charged substrate, oxaloacetate, binds near the base of this helix in the large domain and the interaction of Arg329 at the base of the helix with oxaloacetate is one that is consistent with the shift of this helix in going from the crystallographic open to closed structure. Therefore, the results suggest that without the substrate the enzyme remains in a partially open conformation ready to receive the substrate. In this way, the efficiency of the enzyme should be increased over one that is closed part of the time, with its binding site inaccessible to the substrate.     

The effects of E7 and E11 mutations on the kinetics of ligand binding to R state human hemoglobin

Association and dissociation rate constants for O2, CO, and methyl isocyanide binding to native and distal pocket mutants of R state human hemoglobin were measured using ligand displacement and partial photolysis techniques. Individual rate constants for the alpha and beta subunits were resolved by comparisons between the kinetic behavior of the native and mutant proteins. His-E7 was replaced with Gly and Gln in both alpha and beta subunits and with Phe in beta subunits alone. In separate experiments Val-E11 was replaced with Ala, Leu, and Ile in each globin chain. The parameters describing ligand binding to R state alpha subunits are sensitive to the size and polarity of the amino acids at positions E7 and E11. The distal histidine in this subunit inhibits the bimolecular rate of binding of both O2 and CO, sterically hinders bound CO and methyl isocyanide, and stabilizes bound O2 by hydrogen bonding. The Val-E11 side chain in alpha chains also appears to be part of the kinetic barrier to O2 and CO binding since substitution with Ala causes approximately 10-fold increases in the association rate constants for the binding of these diatomic ligands. However, substitution of Val-E11 by Ile produces only small decreases in the rates of ligand binding to alpha subunits. For R state beta subunits, the bimolecular rates of O2 and CO binding are intrinsically large, approximately 2-5-fold greater than those for alpha subunits, and with the exception of Val-E11----Ile mutation, little affected by substitutions at either the E7 or E11 positions. For the beta Val-E11----Ile mutant the association rate and equilibrium constants for all three ligands decreased 10-50-fold. All of these results agree with Shaanan's conclusions that the distal pocket in liganded beta subunits is more open whereas in alpha subunits bound ligands are more sterically hindered by adjacent distal residues (Shaanan, B. (1983) J. Mol. Biol. 171, 31-59). In the case of O2 binding to alpha subunits, the unfavorable steric effects are compensated by the formation of a hydrogen bond between the nitrogen atom of His-E7 and bound dioxygen.     

Structural basis for HNF-4alpha activation by ligand and coactivator binding

In addition to suggesting that fatty acids are endogenous ligands, our recent crystal structure of HNF-4alpha showed an unusual degree of structural flexibility in the AF-2 domain (helix alpha12). Although every molecule contained a fatty acid within its ligand binding domain, one molecule in each homodimer was in an open inactive conformation with alpha12 fully extended and colinear with alpha10. By contrast, the second molecule in each homodimer was in a closed conformation with alpha12 folded against the body of the domain in what is widely considered to be the active state. This indicates that although ligand binding is necessary, it is not sufficient to induce an activating structural transition in HNF-4alpha as is commonly suggested to occur for nuclear receptors. To further assess potential mechanisms of activation, we have solved a structure of human HNF-4alpha bound to both fatty acid ligand and a coactivator sequence derived from SRC-1. The mode of coactivator binding is similar to that observed for other nuclear receptors, and in this case, all of the molecules adopt the closed active conformation. We conclude that for HNF-4alpha, coactivator rather than ligand binding locks the active conformation.     

Change of pore helix conformational state upon opening of cyclic nucleotide-gated channels

The structure of the pore region of the alpha subunit of the bovine rod cyclic nucleotide-gated channel was probed using cysteine-scanning mutagenesis and hydrophilic sulfhydryl-reactive methanethiosulfonate (MTS) reagents. A region homologous to the pore helix in the X-ray crystal structure of the KcsA K(+) channel showed a helical pattern of reactivity with externally applied MTS reagents. Surprisingly, three out of four of the reactive residues, all on one face of the pore helix, only reacted with MTS reagents in the closed state. A residue on the opposite face of the helix only reacted with MTS reagents in the open state. These results indicate that the pore helix (or its surroundings) undergoes a change in conformation, perhaps involving a rotation around its long axis, that opens a gate localized to the selectivity filter of the channel.