Blocking the gate to ligand entry in human hemoglobin.
ABSTRACT: His(E7) to Trp replacements in HbA lead to markedly biphasic bimolecular CO rebinding after laser photolysis. For isolated mutant subunits, the fraction of fast phase increases with increasing [CO], suggesting a competition between binding to an open conformation with an empty E7 channel and relaxation to blocked or closed, slowly reacting states. The rate of conformational relaxation of the open state is ∼18,000 s(-1) in α subunits and ∼10-fold faster in β subunits, ∼175,000 s(-1). Crystal structures were determined for tetrameric α(WT)β(Trp-63) HbCO, α(Trp-58)β(WT) deoxyHb, and Trp-64 deoxy- and CO-Mb as controls. In Trp-63(E7) βCO, the indole side chain is located in the solvent interface, blocking entry into the E7 channel. Similar blocked Trp-64(E7) conformations are observed in the mutant Mb crystal structures. In Trp-58(E7) deoxy-α subunits, the indole side chain fills both the channel and the distal pocket, forming a completely closed state. The bimolecular rate constant for CO binding, k'(CO), to the open conformations of both mutant Hb subunits is ∼80-90 μm(-1) s(-1), whereas k'(CO) for the completely closed states is 1000-fold slower, ∼0.08 μm(-1) s(-1). A transient intermediate with k'(CO) ≈ 0.7 μm(-1) s(-1) is observed after photolysis of Trp-63(E7) βCO subunits and indicates that the indole ring blocks the entrance to the E7 channel, as observed in the crystal structures of Trp(E7) deoxyMb and βCO subunits. Thus, either blocking or completely filling the E7 channel dramatically slows bimolecular binding, providing strong evidence that the E7 channel is the major pathway (≥90%) for ligand entry in human hemoglobin.
Project description:The role of the distal histidine in regulating ligand binding to adult human hemoglobin (HbA) was re-examined systematically by preparing His(E7) to Gly, Ala, Leu, Gln, Phe, and Trp mutants of both Hb subunits. Rate constants for O(2), CO, and NO binding were measured using rapid mixing and laser photolysis experiments designed to minimize autoxidation of the unstable apolar E7 mutants. Replacing His(E7) with Gly, Ala, Leu, or Phe causes 20-500-fold increases in the rates of O(2) dissociation from either Hb subunit, demonstrating unambiguously that the native His(E7) imidazole side chain forms a strong hydrogen bond with bound O(2) in both the alpha and beta chains (DeltaG(His(E7)H-bond) approximately -8 kJ/mol). As the size of the E7 amino acid is increased from Gly to Phe, decreases in k(O2)', k(NO)', and calculated bimolecular rates of CO entry (k(entry)') are observed. Replacing His(E7) with Trp causes further decreases in k(O2)', k(NO)', and k(entry)' to 1-2 microM(-1) s(-1) in beta subunits, whereas ligand rebinding to alphaTrp(E7) subunits after photolysis is markedly biphasic, with fast k(O2)', k(CO)', and k(NO)' values approximately 150 microM(-1) s(-1) and slow rate constants approximately 0.1 to 1 microM(-1) s(-1). Rapid bimolecular rebinding to an open alpha subunit conformation occurs immediately after photolysis of the alphaTrp(E7) mutant at high ligand concentrations. However, at equilibrium the closed alphaTrp(E7) side chain inhibits the rate of ligand binding >200-fold. These data suggest strongly that the E7 side chain functions as a gate for ligand entry in both HbA subunits.
Project description:The free volume in the active site of human HbA plays a crucial role in governing the bimolecular rates of O(2), CO, and NO binding, the fraction of geminate ligand recombination, and the rate of NO dioxygenation by the oxygenated complex. We have decreased the size of the distal pocket by mutating Leu(B10), Val(E11), and Leu(G8) to Phe and Trp and that of other more internal cavities by filling them with Xe at high gas pressures. Increasing the size of the B10 side chain reduces bimolecular rates of ligand binding nearly 5000-fold and inhibits CO geminate recombination due to both reduction of the capture volume in the distal pocket and direct steric hindrance of Fe-ligand bond formation. Phe and Trp(E11) mutations also cause a decrease in distal pocket volume but, at the same time, increase access to the Fe atom because of the loss of the ?2 CH(3) group of the native Val(E11) side chain. The net result of these E11 substitutions is a dramatic increase in the rate of geminate recombination because dissociated CO is sequestered close to the Fe atom and can rapidly rebind without steric resistance. However, the bimolecular rate constants for binding of ligand to the Phe and Trp(E11) mutants are decreased 5-30-fold, because of a smaller capture volume. Geminate and bimolecular kinetic parameters for Phe and Trp(G8) mutants are similar to those for the native HbA subunits because the aromatic rings at this position cause little change in distal pocket volume and because ligands do not move past this position into the globin interior of wild-type HbA subunits. The latter conclusion is verified by the observation that Xe binding to the ? and ? Hb subunits has little effect on either geminate or bimolecular ligand rebinding. All of these experimental results argue strongly against alternative ligand migration pathways that involve movements through the protein interior in HbA. Instead, ligands appear to enter through the His(E7) gate and are captured directly in the distal cavity.
Project description:The major pathway for O2 binding to mammalian myoglobins (Mb) and hemoglobins (Hb) involves transient upward movement of the distal histidine (His-64(E7)), allowing ligand capture in the distal pocket. The mini-globin from Cerebratulus lacteus (CerHb) appears to have an alternative pathway between the E and H helices that is made accessible by loss of the N-terminal A helix. To test this pathway, we examined the effects of changing the size of the E7 gate and closing the end of the apolar channel in CerHb by site-directed mutagenesis. Increasing the size of Gln-44(E7) from Ala to Trp causes variation of association (k'O2) and dissociation (kO2) rate coefficients, but the changes are not systematic. More significantly, the fractions (Fgem approximately 0.05-0.19) and rates (kgem approximately 50-100 micros(-1)) of geminate CO recombination in the Gln-44(E7) mutants are all similar. In contrast, blocking the entrance to the apolar channel by increasing the size of Ala-55(E18) to Phe and Trp causes the following: 1) both k'O2 and kO2 to decrease roughly 4-fold; 2) Fgem for CO to increase from approximately 0.05 to 0.45; and 3) kgem to decrease from approximately 80 to approximately 9 micros(-1), as ligands become trapped in the channel. Crystal structures and low temperature Fourier-transform infrared spectra of Phe-55 and Trp-55 CerHb confirm that the aromatic side chains block the channel entrance, with little effect on the distal pocket. These results provide unambiguous experimental proof that diatomic ligands can enter and exit a globin through an interior channel in preference to the more direct E7 pathway.
Project description:Since the elucidation of the myoglobin (Mb) structure, a histidine residue on the E helix (His-E7) has been proposed to act as a gate with an open or closed conformation controlling access to the active site. Although it is believed that at low pH, the His-E7 gate is in its open conformation, the full relationship between the His-E7 protonation state, its conformation, and ligand migration in Mb is hotly debated. We used molecular dynamics simulations to first address the effect of His-E7 protonation on its conformation. We observed the expected shift from the closed to the open conformation upon protonation, but more importantly, noted a significant difference between the conformations of the two neutral histidine tautomers. We further computed free energy profiles for oxygen migration in each of the possible His-E7 states as well as in two instructive Mb mutants: Ala-E7 and Trp-E7. Our results show that even in the closed conformation, the His-E7 gate does not create a large barrier to oxygen migration and permits oxygen entry with only a small rotation of the imidazole side chain and movement of the E helix. We identify, instead, a hydrophobic site in the E7 channel that can accommodate an apolar diatomic ligand and enhances ligand uptake particularly in the open His-E7 conformation. This rate enhancement is diminished in the closed conformation. Taken together, our results provide a new conceptual framework for the histidine gate hypothesis.
Project description:The large-conductance Ca(2+)-activated K(+) (BK(Ca)) channel is essential for maintaining the membrane in a hyperpolarized state, thereby regulating neuronal excitability, smooth muscle contraction, and secretion. The BK(Ca) α-subunit has three predicted initiation codons that generate proteins with N-terminal ends starting with the amino acid sequences MANG, MSSN, or MDAL. Because the N-terminal region and first transmembrane domain of the α-subunit are required for modulation by auxiliary β1-subunits, we examined whether β1 differentially modulates the N-terminal BK(Ca) α-subunit isoforms. In the absence of β1, all isoforms had similar single-channel conductances and voltage-dependent activation. However, whereas β1 did not modulate the voltage-activation curve of MSSN, β1 induced a significant leftward shift of the voltage activation curves of both the MDAL and MANG isoforms. These shifts, of which the MDAL was larger, occurred at both 10 μM and 100 μM Ca(2+). The β1-subunit increased the open dwell times of all three isoforms and decreased the closed dwell times of MANG and MDAL but increased the closed dwell times of MSSN. The distinct modulation of voltage activation by the β1-subunit may be due to the differential effect of β1 on burst duration and interburst intervals observed among these isoforms. Additionally, we observed that the related β2-subunit induced comparable leftward shifts in the voltage-activation curves of all three isoforms, indicating that the differential modulation of these isoforms was specific to β1. These findings suggest that the relative expression of the N-terminal isoforms can fine-tune BK(Ca) channel activity in cells, highlighting a novel mechanism of BK(Ca) channel regulation.
Project description:The monovalent cation (MVC) site of the tryptophan synthase from Salmonella typhimurium plays essential roles in catalysis and in the regulation of substrate channeling. In vitro, MVCs affect the equilibrium distribution of intermediates formed in the reaction of l-Ser with the alpha(2)beta(2) complex; the MVC-free, Cs(+)-bound, and NH(4)(+)-bound enzymes stabilize the alpha-aminoacrylate species, E(A-A), while Na(+) binding stabilizes the l-Ser external aldimine species, E(Aex(1)). Two probes of beta-site reactivity and conformation were used herein, the reactive indole analogue, indoline, and the l-Trp analogue, l-His. MVC-bound E(A-A) reacts rapidly with indoline to give the indoline quinonoid species, E(Q)(indoline), which slowly converts to dihydroiso-l-tryptophan. MVC-free E(A-A) gives very little E(Q)(indoline), and turnover is strongly impaired; the fraction of E(Q)(indoline) formed is <3.5% of that given by the Na(+)-bound form. The reaction of l-Ser with the MVC-free internal aldimine species, E(Ain), initially gives small amounts of an active E(A-A) which converts to an inactive species on a slower, conformational, time scale. This inactivation is abolished by the binding of MVCs. The inactive E(A-A) appears to have a closed beta-subunit conformation with an altered substrate binding site that is different from the known conformations of tryptophan synthase. Reaction of l-His with E(Ain) gives an equilibrating mixture of external aldimine and quinonoid species, E(Aex)(his) and E(Q)(his). The MVC-free and Na(+) forms of the enzyme gave trace amounts of E(Q)(his) ( approximately 1% of the beta-sites). The Cs(+) and NH(4)(+) forms gave approximately 17 and approximately 14%, respectively. The reactivity of MVC-free E(Ain) was restored by the binding of an alpha-site ligand. These studies show MVCs and alpha-site ligands act synergistically to modulate the switching of the beta-subunit from the open to the closed conformation, and this switching is crucial to the regulation of beta-site catalytic activity. Comparison of the structures of Na(+) and Cs(+) forms of the enzyme shows Cs(+) favors complexes with open indole binding sites poised for the conformational transition to the closed state, whereas the Na(+) form does not. The beta-subunits of Cs(+) complexes exhibit preformed indole subsites; the indole subsites of the open Na(+) complexes are collapsed, distorted, and too small to accommodate indole.
Project description:G-protein betagamma (Gbetagamma) subunits interact with a wide range of molecular partners including: G(alpha) subunits, effectors, peptides, and small molecule inhibitors. The molecular mechanisms underlying the ability to accommodate this wide range of structurally distinct binding partners are not well understood. To uncover the role of protein flexibility and alterations in protein conformation in molecular recognition by Gbetagamma, a method for site-specific (15)N-labeling of Gbeta-Trp residue backbone and indole amines in insect cells was developed. Transverse Relaxation Optimized Spectroscopy-Heteronuclear Single-Quantum Coherence Nuclear Magnetic Resonance (TROSY-HSQC NMR) analysis of (15)N-Trp Gbetagamma identified well-dispersed signals for the individual Trp residue side chain and amide positions. Surprisingly, a wide range of signal intensities was observed in the spectrum, likely representing a range of backbone and side chain mobilities. The signal for GbetaW99 indole was very intense, suggesting a high level of mobility on the protein surface and molecular dynamics simulations indicate that GbetaW99 is highly mobile on the nanosecond timescale in comparison with other Gbeta tryptophans. Binding of peptides and phosducin dramatically altered the mobility of GbetaW99 and GbetaW332 in the binding site and the chemical shifts at sites distant from the direct binding surface in distinct ways. In contrast, binding of G(alpha)(i1)-GDP to Gbetagamma had relatively little effect on the spectrum and, most surprisingly, did not significantly alter Trp mobility at the subunit interface. This suggests the inactive heterotrimer in solution adopts a conformation with an open subunit interface a large percentage of the time. Overall, these data show that Gbetagamma subunits explore a range of conformations that can be exploited during molecular recognition by diverse binding partners.
Project description:An adequate response of a living cell to the ever-changing environment requires integration of numerous sensory inputs. In many cases, it can be achieved even at the level of a single receptor molecule. Polymodal transient receptor potential (TRP) channels have been shown to integrate mechanical, chemical, electric, and thermal stimuli. Inappropriate gating can lead to pathologies. Among the >60 known TRP vanilloid subfamily (V) 4 mutations that interfere with bone development are Y602C or R616Q at the S4-S5 linker. A cation-? bond between the conservative residues Y602 and R616 of neighboring subunits appears likely in many homologous channel structures in a closed state. Our experiments with TRPV4 mutants indicate that the resting-closed state remains stable while the bond is substituted by a salt bridge or disulfide bond, whereas disruption of the contact by mutations like Y602C or R616Q produces gain-of-function phenotypes when TRPV4 is heterologously expressed in the Xenopus oocyte or yeast. Our data indicate that the Y602-R616 cation-? interactions link the four S4-S5 linker helices together, forming a girdle backing the closed gate. Analogous cation-? bonds and the girdle are seen in many closed TRP channel structures. This girdle is not observed in the cryo-EM structure of amphibian TRPV4 (Protein Data Bank ID code 6BBJ), which appears to be in a different impermeable state-we hypothesize this is the inactivated state.
Project description:The large apolar tunnel traversing the mini-hemoglobin from Cerebratulus lacteus (CerHb) has been examined by x-ray crystallography, ligand binding kinetics, and molecular dynamic simulations. The addition of 10 atm of xenon causes loss of diffraction in wild-type (wt) CerHbO(2) crystals, but Leu-86(G12)Ala CerHbO(2), which has an increased tunnel volume, stably accommodates two discrete xenon atoms: one adjacent to Leu-86(G12) and another near Ala-55(E18). Molecular dynamics simulations of ligand migration in wt CerHb show a low energy pathway through the apolar tunnel when Leu or Ala, but not Phe or Trp, is present at the 86(G12) position. The addition of 10-15 atm of xenon to solutions of wt CerHbCO and L86A CerHbCO causes 2-3-fold increases in the fraction of geminate ligand recombination, indicating that the bound xenon blocks CO escape. This idea was confirmed by L86F and L86W mutations, which cause even larger increases in the fraction of geminate CO rebinding, 2-5-fold decreases in the bimolecular rate constants for ligand entry, and large increases in the computed energy barriers for ligand movement through the apolar tunnel. Both the addition of xenon to the L86A mutant and oxidation of wt CerHb heme iron cause the appearance of an out Gln-44(E7) conformer, in which the amide side chain points out toward the solvent and appears to lower the barrier for ligand escape through the E7 gate. However, the observed kinetics suggest little entry and escape (? 25%) through the E7 pathway, presumably because the in Gln-44(E7) conformer is thermodynamically favored.
Project description:Coiled-coil motifs are ubiquitous mediators of specific protein-protein interactions through the formation of interlocking hydrophobic seams between alpha-helical chains. Residues that form these seams occur at the first (a) and fourth (d) positions of a characteristic 7-aa repeat and are primarily aliphatic. The potential of aromatic residues to promote helix association in a coiled coil was explored by engineering a "Trp-zipper" protein with Trp residues at all 14 a and d positions. The protein forms a discrete, stable, alpha-helical pentamer in water at physiological pH. Its 1.45-A crystal structure reveals a parallel, five-stranded coiled coil, a previously uncharacterized type of "knobs-into-holes" packing interaction between interfacial Trp side chains, and an unusual approximately 8-A-diameter axial channel lined with indole rings that is filled with polyethylene glycol 400 and water and sulfate ion molecules. The engineered Trp-zipper pentamer enlarges current views of coiled-coil assembly, molecular recognition, and protein engineering, and may serve as a soluble model for membrane ion channels.