The Functional Roles of the His247 and His281 Residues in Folate and Proton Translocation Mediated by the Human Proton-coupled Folate Transporter SLC46A1

This report addresses the functional role of His residues in the proton-coupled folate transporter (PCFT; SLC46A1), which mediates intestinal folate absorption. Of ten His residues, only H247A and H281A mutations altered function. The folic acid influx K_t at pH 5.5 for H247A was ↓8.4-fold. Although wild type (WT)-PCFT K_i values varied among the folates, K_i values were much lower and comparable for H247-A, -R, -Q, or -E mutants. Homology modeling localized His²⁴⁷ to the large loop separating transmembrane domains 6 and 7 at the cytoplasmic entrance of the translocation pathway in hydrogen-bond distance to Ser¹⁷². The folic acid influx K_t for S172A-PCFT was decreased similar to H247A. His²⁸¹ faces the extracellular region in the seventh transmembrane domain. H281A-PCFT results in loss-of-function due to ∼12-fold↑ in the folic acid influx K_t. When the pH was decreased from 5.5 to 4.5, the WT-PCFT folic acid influx K_t was unchanged, but the K_t decreased 4-fold for H281A. In electrophysiological studies in Xenopus oocytes, both WT-PCFT- and H281A-PCFT-mediated folic acid uptake produced current and acidification, and both exhibited a low level of folate-independent proton transport (slippage). Slippage was markedly increased for the H247A-PCFT mutant. The data suggest that disruption of the His²⁴⁷ to Ser¹⁷² interaction results in a PCFT conformational alteration causing a loss of selectivity, increased substrate access to a high affinity binding pocket, and proton transport in the absence of a folate gradient. The His²⁸¹ residue is not essential for proton coupling but plays an important role in PCFT protonation, which, in turn, augments folate binding to the carrier.     

Solution structure of human von Willebrand factor studied using small angle neutron scattering

von Willebrand factor (VWF) binding to platelets under high fluid shear is an important step regulating atherothrombosis. We applied light and small angle neutron scattering to study the solution structure of human VWF multimers and protomer. Results suggest that these proteins resemble prolate ellipsoids with radius of gyration (R(g)) of approximately 75 and approximately 30 nm for multimer and protomer, respectively. The ellipsoid dimensions/radii are 175 x 28 nm for multimers and 70 x 9.1 nm for protomers. Substructural repeat domains are evident within multimeric VWF that are indicative of elements of the protomer quarternary structure (16 nm) and individual functional domains (4.5 nm). Amino acids occupy only approximately 2% of the multimer and protomer volume, compared with 98% for serum albumin and 35% for fibrinogen. VWF treatment with guanidine.HCl, which increases VWF susceptibility to proteolysis by ADAMTS-13, causes local structural changes at length scales <10 nm without altering protein R(g). Treatment of multimer but not protomer VWF with random homobifunctional linker BS(3) prior to reduction of intermonomer disulfide linkages and Western blotting reveals a pattern of dimer and trimer units that indicate the presence of stable intermonomer non-covalent interactions within the multimer. Overall, multimeric VWF appears to be a loosely packed ellipsoidal protein with non-covalent interactions between different monomer units stabilizing its solution structure. Local, and not large scale, changes in multimer conformation are sufficient for ADAMTS-13-mediated proteolysis.     

Allosteric HIV‐1 integrase inhibitors promote aberrant protein multimerization by directly mediating inter‐subunit interactions: Structural and thermodynamic modeling studies

Allosteric HIV‐1 integrase (IN) inhibitors (ALLINIs) bind at the dimer interface of the IN catalytic core domain (CCD), and potently inhibit HIV‐1 by promoting aberrant, higher‐order IN multimerization. Little is known about the structural organization of the inhibitor‐induced IN multimers and important questions regarding how ALLINIs promote aberrant IN multimerization remain to be answered. On the basis of physical chemistry principles and from our analysis of experimental information, we propose that inhibitor‐induced multimerization is mediated by ALLINIs directly promoting inter‐subunit interactions between the CCD dimer and a C‐terminal domain (CTD) of another IN dimer. Guided by this hypothesis, we have built atomic models of inter‐subunit interfaces in IN multimers by incorporating information from hydrogen‐deuterium exchange (HDX) measurements to drive protein‐protein docking. We have also developed a novel free energy simulation method to estimate the effects of ALLINI binding on the association of the CCD and CTD. Using this structural and thermodynamic modeling approach, we show that multimer inter‐subunit interface models can account for several experimental observations about ALLINI‐induced multimerization, including large differences in the potencies of various ALLINIs, the mechanisms of resistance mutations, and the crucial role of solvent exposed R‐groups in the high potency of certain ALLINIs. Our study predicts that CTD residues Tyr226, Trp235 and Lys266 are involved in the aberrant multimer interfaces. The key finding of the study is that it suggests the possibility of ALLINIs facilitating inter‐subunit interactions between an external CTD and the CCD‐CCD dimer interface.     

A pH-regulated dimeric bouquet in the structure of von Willebrand factor

At the acidic pH of the trans-Golgi and Weibel-Palade bodies (WPBs), but not at the alkaline pH of secretion, the C-terminal ～1350 residues of von Willebrand factor (VWF) zip up into an elongated, dimeric bouquet. Six small domains visualized here for the first time between the D4 and cystine-knot domains form a stem. The A2, A3, and D4 domains form a raceme with three pairs of opposed, large, flower-like domains. N-terminal VWF domains mediate helical tubule formation in WPBs and template N-terminal disulphide linkage between VWF dimers, to form ultralong VWF concatamers. The dimensions we measure in VWF at pH 6.2 and 7.4, and the distance between tubules in nascent WPB, suggest that dimeric bouquets are essential for correct VWF dimer incorporation into growing tubules and to prevent crosslinking between neighbouring tubules. Further insights into the structure of the domains and flexible segments in VWF provide an overall view of VWF structure important for understanding both the biogenesis of ultralong concatamers at acidic pH and flow-regulated changes in concatamer conformation in plasma at alkaline pH that trigger hemostasis.     

Activation and Proton Transport Mechanism in Influenza A M2 Channel

Molecular dynamics trajectories 2 μs in length have been generated for the pH-activated, tetrameric M2 proton channel of the influenza A virus in all protonation states of the pH sensor located at the His³⁷ tetrad. All simulated structures are in very good agreement with high-resolution structures. Changes in the channel caused by progressive protonation of His³⁷ provide insight into the mechanism of proton transport. The channel is closed at both His³⁷ and Trp⁴¹ sites in the singly and doubly protonated states, but it opens at Trp⁴¹ upon further protonation. Anions access the charged His³⁷ and by doing so stabilize the protonated states of the channel. The narrow opening at the His³⁷ site, further blocked by anions, is inconsistent with the water-wire mechanism of proton transport. Instead, conformational interconversions of His³⁷ correlated with hydrogen bonding to water molecules indicate that these residues shuttle protons in high-protonation states. Hydrogen bonds between charged and uncharged histidines are rare. The valve at Val²⁷ remains on average quite narrow in all protonation states but fluctuates sufficiently to support water and proton transport. A proton transport mechanism in which the channel, depending on pH, opens at either the histidine or valine gate is only partially supported by the simulations.     

Activation and Proton Transport Mechanism in Influenza A M2 Channel

Molecular dynamics trajectories 2 μs in length have been generated for the pH-activated, tetrameric M2 proton channel of the influenza A virus in all protonation states of the pH sensor located at the His³⁷ tetrad. All simulated structures are in very good agreement with high-resolution structures. Changes in the channel caused by progressive protonation of His³⁷ provide insight into the mechanism of proton transport. The channel is closed at both His³⁷ and Trp⁴¹ sites in the singly and doubly protonated states, but it opens at Trp⁴¹ upon further protonation. Anions access the charged His³⁷ and by doing so stabilize the protonated states of the channel. The narrow opening at the His³⁷ site, further blocked by anions, is inconsistent with the water-wire mechanism of proton transport. Instead, conformational interconversions of His³⁷ correlated with hydrogen bonding to water molecules indicate that these residues shuttle protons in high-protonation states. Hydrogen bonds between charged and uncharged histidines are rare. The valve at Val²⁷ remains on average quite narrow in all protonation states but fluctuates sufficiently to support water and proton transport. A proton transport mechanism in which the channel, depending on pH, opens at either the histidine or valine gate is only partially supported by the simulations.     

Tyrosyl-DNA Phosphodiesterase I Catalytic Mutants Reveal an Alternative Nucleophile That Can Catalyze Substrate Cleavage

Tyrosyl-DNA phosphodiesterase I (Tdp1) catalyzes the repair of 3′-DNA adducts, such as the 3′-phosphotyrosyl linkage of DNA topoisomerase I to DNA. Tdp1 contains two conserved catalytic histidines: a nucleophilic His (His^nuc) that attacks DNA adducts to form a covalent 3′-phosphohistidyl intermediate and a general acid/base His (His^gab), which resolves the Tdp1-DNA linkage. A His^nuc to Ala mutant protein is reportedly inactive, whereas the autosomal recessive neurodegenerative disease SCAN1 has been attributed to the enhanced stability of the Tdp1-DNA intermediate induced by mutation of His^gab to Arg. However, here we report that expression of the yeast His^nucAla (H182A) mutant actually induced topoisomerase I-dependent cytotoxicity and further enhanced the cytotoxicity of Tdp1 His^gab mutants, including H432N and the SCAN1-related H432R. Moreover, the His^nucAla mutant was catalytically active in vitro, albeit at levels 85-fold less than that observed with wild type Tdp1. In contrast, the His^nucPhe mutant was catalytically inactive and suppressed His^gab mutant-induced toxicity. These data suggest that the activity of another nucleophile when His^nuc is replaced with residues containing a small side chain (Ala, Asn, and Gln), but not with a bulky side chain. Indeed, genetic, biochemical, and mass spectrometry analyses show that a highly conserved His, immediately N-terminal to His^nuc, can act as a nucleophile to catalyze the formation of a covalent Tdp1-DNA intermediate. These findings suggest that the flexibility of Tdp1 active site residues may impair the resolution of mutant Tdp1 covalent phosphohistidyl intermediates and provide the rationale for developing chemotherapeutics that stabilize the covalent Tdp1-DNA intermediate.     

Shear-induced disulfide bond formation regulates adhesion activity of von Willebrand factor

von Willebrand factor (VWF) is the largest multimeric adhesion ligand circulating in blood. Its adhesion activity is related to multimer size, with the ultra-large forms freshly released from the activated endothelial cells being most active, capable of spontaneously binding to platelets. In comparison, smaller plasma forms circulating in blood bind platelets only under high fluid shear stress or induced by modulators. The structure-function relationships that distinguish the two types of VWF multimers are not known. In this study, we demonstrate that some of the plasma VWF multimers contain surface-exposed free thiols. Physiological and pathological levels of shear stresses (50 and 100 dynes/cm(2)) promote the formation of disulfide bonds utilizing these free thiols. The shear-induced thiol-disulfide exchange increases VWF binding to platelets. The thiol-disulfide exchange involves some or all of nine cysteine residues (Cys(889), Cys(898), Cys(2448), Cys(2451), Cys(2490), Cys(2491), Cys(2453), Cys(2528), and Cys(2533)) in the D3 and C domains as determined by mass spectrometry of the tryptic VWF peptides. These results suggest that the thiol-disulfide state may serve as an important structural determinant of VWF adhesion activity and can be modified by fluid shear stress.     

Atpase activity and multimer formation of Pilq protein are required for thin pilus biogenesis in plasmid R64

Plasmid R64 pilQ gene is essential for the formation of thin pilus, a type IV pilus. The pilQ product contains NTP binding motifs and belongs to the PulE-VirB11 family of NTPases. The pilQ gene was overexpressed with an N-terminal His tag, and PilQ protein was purified. Purified His tag PilQ protein displayed ATPase activity with a V(max) of 0.71 nmol/min/mg of protein and a K(m) of 0.26 mm at pH 6.5. By gel filtration chromatography, PilQ protein was eluted at the position corresponding to 460 kDa, suggesting that PilQ protein forms a homooctamer. To analyze the relationship between structure and function of PilQ protein, amino acid substitutions were introduced within several conserved motifs. Among 11 missense mutants, 7 mutants exhibited various levels of reduced DNA transfer frequencies in liquid matings. Four mutant genes (T234I, K238Q, D263N, and H328A) were overexpressed with a His tag. The purified mutant PilQ proteins contained various levels of reduced ATPase activity. Three mutant PilQ proteins formed stable multimers similar to wild-type PilQ, whereas the PilQ D263N multimer was unstable. PilQ D263N monomer exhibited low ATPase activity, while PilQ D263N multimer did not. These results indicate that ATPase activity of the PilQ multimer is essential for R64 thin pilus biogenesis.     

A Tyrosine Residue on the TSH Receptor Stabilizes Multimer Formation

Background

The thyrotropin stimulating hormone receptor (TSHR) is a G protein coupled receptor (GPCR) with a large ectodomain. The ligand, TSH, acting via this receptor regulates thyroid growth and thyroid hormone production and secretion. The TSH receptor (TSHR) undergoes complex post –translational modifications including intramolecular cleavage and receptor multimerization. Since monomeric and multimeric receptors coexist in cells, understanding the functional role of just the TSHR multimers is difficult. Therefore, to help understand the physiological significance of receptor multimerization, it will be necessary to abrogate multimer formation, which requires identifying the ectodomain and endodomain interaction sites on the TSHR. Here, we have examined the contribution of the ectodomain to constitutive multimerization of the TSHR and determined the possible residue(s) that may be involved in this interaction.

Methodology/Principal Findings

We studied ectodomain multimer formation by expressing the extracellular domain of the TSHR linked to a glycophosphotidyl (GPI) anchor in both stable and transient expression systems. Using co-immunoprecipitation and FRET of tagged receptors, we established that the TSH receptor ectodomain was capable of multimerization even when totally devoid of the transmembrane domain. Further, we studied the effect of two residues that likely made critical contact points in this interaction. We showed that a conserved tyrosine residue (Y116) on the convex surface of the LRR3 was a critical residue in ectodomain multimer formation since mutation of this residue to serine totally abrogated ectodomain multimers. This abrogation was not seen with the mutation of cysteine 176 on the inner side of the LRR5, demonstrating that inter-receptor disulfide bonding was not involved in ectodomain multimer formation. Additionally, the Y116 mutation in the intact wild type receptor enhanced receptor degradation.

Conclusions/Significance

These data establish the TSH receptor ectodomain as one site of multimerization, independent of the transmembrane region, and that this interaction was primarily via a conserved tyrosine residue in LRR3.     

Structural hierarchy of mechanical extensibility in human von Willebrand factor multimers

The von Willebrand factor (VWF) is a multimeric glycoprotein composed of 80- to 120-nm-long protomeric units and plays a fundamental role in mediating platelet function at high shear. The exact nature of the shear-induced structural transitions have remained elusive; uncovering them requires the high-resolution quantitative analysis of gradually extended VWF. Here, we stretched human blood-plasma-derived VWF with molecular combing and analyzed the axial structure of the elongated multimers with atomic force microscopy. Protomers extended through structural intermediates that could be grouped into seven distinct topographical classes. Protomer extension thus progresses through the uncoiling of the C_1–6 domain segment, rearrangements among the N-terminal VWF domains, and unfolding and elastic extension of the A₂ domain. The least and most extended protomer conformations were localized at the ends and the middle of the multimer, respectively, revealing an apparent necking phenomenon characteristic of plastic-material behavior. The structural hierarchy uncovered here is likely to provide a spatial control mechanism to the complex functions of VWF.     

Domains involved in multimer assembly of von willebrand factor (vWF): multimerization is independent of dimerization.

The precursor protein of von Willebrand factor (pro-vWF) consist of four repeated domains, denoted D1-D2-D'-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2. The domains D1 and D2 constitute the amino-terminal pro-polypeptide and the remaining domains mature vWF, generated upon proteolytic processing. We have shown previously that the pro-polypeptide of pro-vWF is obligatory for assembly of pro-vWF dimers into multimers, a process vital for efficient adhesion of platelets to an injured vessel wall. Here, we have employed full length vWF cDNA to construct a series of deletion mutants, based on the homology between the various domains. Specifically, the domains D', D3 or both were deleted and the multimeric pattern of the mutant vWF proteins was analysed after transient expression in COS-1 cells. It is demonstrated that in addition to the pro-polypeptide, both the D' and the D3 domain are required for multimer assembly. Furthermore, by analysing a construct containing only the domains D' and D3 next to the pro-polypeptide it is shown that this is the only part of the vWF protein involved in multimer assembly. Since, the formation of pro-vWF dimers relies on the carboxy-terminal area of mature vWF, it is concluded that multimer assembly is a process independent of dimerization.     

ExeA binds to peptidoglycan and forms a multimer for assembly of the type II secretion apparatus in Aeromonas hydrophila

Aeromonas hydrophila uses the type II secretion system (T2SS) to transport protein toxins across the outer membrane. The inner membrane complex ExeAB is required for assembly of the ExeD secretion channel multimer, called the secretin, into the outer membrane. A putative peptidoglycan‐binding domain (Pfam number PF01471) conserved in many peptidoglycan‐related proteins is present in the periplasmic region of ExeA (P‐ExeA). In this study, co‐sedimentation analysis revealed that P‐ExeA was able to bind to highly pure peptidoglycan. The protein assembled into large multimers in the presence of peptidoglycan fragments, as shown in native PAGE, gel filtration and cross‐linking experiments. The requirement of peptidoglycan for multimerization was abrogated when the protein was incubated at 30°C and above. These results provide evidence that the putative peptidoglycan‐binding domain of ExeA is involved in physical contact with peptidoglycan. The interactions facilitate the multimerization of ExeA, favouring a model in which the protein forms a multimeric structure on the peptidoglycan during the ExeAB‐dependent assembly of the secretin multimer in the outer membrane.     

Unfolding the A2 Domain of Von Willebrand Factor with the Optical Trap

Von Willebrand factor (VWF) is a multimeric plasma glycoprotein involved in both hemostasis and thrombosis. VWF conformational changes, especially unfolding of the A2 domain, may be required for efficient enzymatic cleavage in vivo. It has been shown that a single A2 domain unfolds at most probable unfolding forces of 7-14 pN at force loading rates of 0.35-350 pN/s and A2 unfolding facilitates A2 cleavage in vitro. However, it remains unknown how much force is required to unfold the A2 domain in the context of a VWF multimer where A2 may be stabilized by other domains like A1 and A3. With the optical trap, we stretched VWF multimers and a poly-protein (A1A2A3)₃ that contains three repeats of the triplet A1A2A3 domains at constant speeds of 2000 nm/s and 400 nm/s, respectively, which yielded corresponding average force loading rates of 90 and 22 pN/s. We found that VWF multimers became stiffer when they were stretched and extended by force. After force increased to a certain level, sudden extensional jumps that signify domain unfolding were often observed. Histograms of the unfolding force and the unfolded contour length showed two or three peaks that were integral multiples of ∼21 pN and ∼63 nm, respectively. Stretching of (A1A2A3)₃ yielded comparable distributions of unfolding force and unfolded contour length, showing that unfolding of the A2 domain accounts for the behavior of VWF multimers under tension. These results show that the A2 domain can be indeed unfolded in the presence of A1, A3, and other domains. Compared with the value in the literature, the larger most probable unfolding force measured in this study suggests that the A2 domain is mechanically stabilized by A1 or A3 although variations in experimental setups and conditions may complicate this interpretation.     

Roles of matrix, p2, and N-terminal myristoylation in human immunodeficiency virus type 1 Gag assembly

Human immunodeficiency virus type 1 Gag protein is cotranslationally myristoylated at the N terminus and targeted to the plasma membrane, where virus particle assembly occurs. Particle assembly requires the ordered multimerization of Gag proteins, yet there is little direct evidence of intermediates of the reaction or of the domains that lead to each stage of the oligomerization process. In this study, following the expression in insect cells of C-terminally truncated Gag proteins and their purification, both the multimeric nature of each Gag protein and the ability to form Gag virus-like particles (VLP) were analyzed. Our results show that (i) the matrix (MA) domain forms a trimer and contributes to a similar level of oligomerization of the assembly-competent Gag; (ii) the p2 domain, located at the capsid/nucleocapsid junction, is essential for a higher order of multimerization (>1,000 kDa); (iii) the latter multimerization is accompanied by a change in Gag assembly morphology from tubes to spheres and results in VLP production; and (iv) N-terminal myristoylation is not required for either of the multimerization stages but plays a key role in conversion of these multimers to Gag VLP. We suggest that the Gag trimer and the > 1,000-kDa multimer are intermediates in the assembly reaction and form before Gag targeting to the plasma membrane. Our data identify a minimum of three stages for VLP development and suggest that each stage involves a separate domain, MA, p2, or N-terminal myristoylation, each of which contributes to HIV particle assembly.     

Cofactor Activity in Factor VIIIa of the Blood Clotting Pathway Is Stabilized by an Interdomain Bond between His281 and Ser524 Formed in Factor VIII

The factor VIII (FVIII) crystal structure suggests a possible bonding interaction of His²⁸¹ (A1 domain) with Ser⁵²⁴ (A2 domain), although the resolution of the structure (∼4 Å) does not firmly establish this bonding. To establish that side chains of these residues participate in an interdomain bond, we prepared and examined the functional properties of a residue swap variant (H281S/S524H) where His²⁸¹ and Ser⁵²⁴ residues were exchanged with one another and a disulfide-bridged variant (H281C/S524C) where the two residues were replaced with Cys. The latter variant showed efficient disulfide bonding of the A1 and A2 domains. The swap variant showed WT-like FVIII and FVIIIa stability, which were markedly reduced for H281A and S524A variants in an earlier study. The disulfide-bridged variant showed ∼20% increased FVIII stability, and FVIIIa did not decay during the time course measured. This variant also yielded 35% increased thrombin peak values compared with WT in a plasma-based thrombin generation assay. Binding analyses of H281S-A1/A3C1C2 dimer with S524H-A2 subunit yielded a near WT-like affinity value, whereas combining the variant dimer or A2 subunit with the WT complement yielded ∼5- and ∼10-fold reductions, respectively, in affinity. Other functional properties including thrombin generation potential, FIXa binding affinity, K_m for FX of FXase complexes, thrombin activation efficiency, and down-regulation by activated protein C showed similar results for the two variants compared with WT FVIII. These results indicate that the side chains of His²⁸¹ and Ser⁵²⁴ are in close proximity and contribute to a bonding interaction in FVIII that is retained in FVIIIa.     

Histidine Residues in the Na+-coupled Ascorbic Acid Transporter-2 (SVCT2) Are Central Regulators of SVCT2 Function,Modulating pH Sensitivity,Transporter Kinetics,Na+ Cooperativity,Conformational Stability,and Subcellular Localization

Na⁺-coupled ascorbic acid transporter-2 (SVCT2) activity is impaired at acid pH, but little is known about the molecular determinants that define the transporter pH sensitivity. SVCT2 contains six histidine residues in its primary sequence, three of which are exofacial in the transporter secondary structure model. We used site-directed mutagenesis and treatment with diethylpyrocarbonate to identify histidine residues responsible for SVCT2 pH sensitivity. We conclude that five histidine residues, His¹⁰⁹, His²⁰³, His²⁰⁶, His²⁶⁹, and His⁴¹³, are central regulators of SVCT2 function, participating to different degrees in modulating pH sensitivity, transporter kinetics, Na⁺ cooperativity, conformational stability, and subcellular localization. Our results are compatible with a model in which (i) a single exofacial histidine residue, His⁴¹³, localized in the exofacial loop IV that connects transmembrane helices VII-VIII defines the pH sensitivity of SVCT2 through a mechanism involving a marked attenuation of the activation by Na⁺ and loss of Na⁺ cooperativity, which leads to a decreased V_max without altering the transport K_m; (ii) exofacial histidine residues His²⁰³, His²⁰⁶, and His⁴¹³ may be involved in maintaining a functional interaction between exofacial loops II and IV and influence the general folding of the transporter; (iii) histidines 203, 206, 269, and 413 affect the transporter kinetics by modulating the apparent transport K_m; and (iv) histidine 109, localized at the center of transmembrane helix I, might be fundamental for the interaction of SVCT2 with the transported substrate ascorbic acid. Thus, histidine residues are central regulators of SVCT2 function.     

External protons destabilize the activated voltage sensor in hERG channels

Extracellular acidosis shifts hERG channel activation to more depolarized potentials and accelerates channel deactivation; however, the mechanisms underlying these effects are unclear. External divalent cations, e.g., Ca²⁺ and Cd²⁺, mimic these effects and coordinate within a metal ion binding pocket composed of three acidic residues in hERG: D456 and D460 in S2 and D509 in S3. A common mechanism may underlie divalent cation and proton effects on hERG gating. Using two-electrode voltage clamp, we show proton sensitivity of hERG channel activation (pKa = 5.6), but not deactivation, was greatly reduced in the presence of Cd²⁺ (0.1 mM), suggesting a common binding site for the Cd²⁺ and proton effect on activation and separable effects of protons on activation and deactivation. Mutational analysis confirmed that D509 plays a critical role in the pH dependence of activation, as shown previously, and that cooperative actions involving D456 and D460 are also required. Importantly, neutralization of all three acidic residues abolished the proton-induced shift of activation, suggesting that the metal ion binding pocket alone accounts for the effects of protons on hERG channel activation. Voltage-clamp fluorimetry measurements demonstrated that protons shifted the voltage dependence of S4 movement to more depolarized potentials. The data indicate a site and mechanism of action for protons on hERG activation gating; protonation of D456, D460 and D509 disrupts interactions between these residues and S4 gating charges to destabilize the activated configuration of S4.     

Interaction between poly(L-lysine48, L-histidine52) and DNA.

Poly(Lys⁴⁸, His⁵²), a random copolypeptide of L -lysine (48%) and L -histidine (52%), was used as a model protein for investigating the effects of protonation on the imidazole group of histidines on protein binding to DNA. The complexes formed between poly(Lys⁴⁸, His⁵²) and DNA were examined using absorbance, circular dichroism (CD), and thermal denaturation. Although increasing pH reduces the charges on histidine side chains in the model protein, the protein still binds the DNA with approximately one positive charge per negative charge in protein-bound regions. Nevertheless, CD and melting properties of poly(Lys⁴⁸, His⁵²)-DNA complexes still depend upon the solution pH which determines the protonation state of imidazole group of histidine side chains. At pH 7.0, the complexes show two characteristic melting bands with a t_m (46–51°C) for free base pairs and a t′_m (94°C) for protein-bound base pairs. The t′_m of the complexes is reduced to 90°C at pH 9.2, although at this pH there is still one lysine per phosphate in protein-bound regions. Presumably, the presence of deprotonated histidine residues destabilizes the native structure of protein-bound DNA. The binding of this model protein to DNA causes a red shift of the crossover point and both a red shift and a reduction of the positive CD band of DNA near 275 nm. This phenomenon is similar to that caused by polylysine binding. These effects, however, are greatly diminished when histidine side chains in the model protein are deprotonated. The structure of already formed poly(Lys⁴⁸, His⁵²)·DNA complexes can be perturbed by changing the solution pH. However, the results suggest a readjustment of the complex to accommodate charge interactions rather than a full dissociation of the complex followed by reassociation between the model protein and DNA.