Effects of charged amino-acid mutation on the solution structure of cytochrome b5 and binding between cytochrome b5 and cytochrome c

The solution structure of oxidized bovine microsomal cytochrome b(5) mutant (E48, E56/A, D60/A) has been determined through 1524 meaningful nuclear Overhauser effect constraints together with 190 pseudocontact shift constraints. The final family of 35 conformers has rmsd values with respect to the mean structure of 0.045+/-0.009 nm and 0.088+/-0.011 nm for backbone and heavy atoms, respectively. A characteristic of this mutant is that of having no significant changes in the whole folding and secondary structure compared with the X-ray and solution structures of wild-type cytochrome b(5). The binding of different surface mutants of cytochrome b(5) with cytochrome c shows that electrostatic interactions play an important role in maintaining the stability and specificity of the protein complex formed. The differences in association constants demonstrate the electrostatic contributions of cytochrome b(5) surface negatively charged residues, which were suggested to be involved in complex formation in the Northrup and Salemme models, have cumulative effect on the stability of cyt c-cyt b(5) complex, and the contribution of Glu48 is a little higher than that of Glu44. Moreover, our result suggests that the docking geometry proposed by Northrup, which is involved in the participation of Glu48, Glu56, Asp60, and heme propionate of cytochrome b(5), do occur in the association between cytochrome b(5) and cytochrome c.     

Solution structure of ZipA, a crucial component of Escherichia coli cell division

ZipA, an essential component of cell division in Escherichia coli, interacts with the FtsZ protein at the midcell in one of the initial steps of septum formation. The high-resolution solution structure of the 144-residue C-terminal domain of E. coli ZipA (ZipA(185)(-)(328)) has been determined by multidimensional heteronuclear NMR. A total of 30 structures were calculated by means of hybrid distance geometry-simulated annealing using a total of 2758 experimental NMR restraints. The atomic root means square distribution about the mean coordinate positions for residues 6-142 for the 30 structures is 0.37 +/- 0.04 A for the backbone atoms, 0. 78 +/- 0.05 A for all atoms, and 0.45 +/- 0.04 A for all atoms excluding disordered side chains. The NMR solution structure of ZipA(185)(-)(328) is composed of three alpha-helices and a beta-sheet consisting of six antiparallel beta-strands where the alpha-helices and the beta-sheet form surfaces directly opposite each other. A C-terminal peptide from FtsZ has been shown to bind ZipA(185)(-)(328) in a hydrophobic channel formed by the beta-sheet providing insight into the ZipA-FtsZ interaction. An unexpected similarity between the ZipA(185)(-)(328) fold and the split beta-alpha-beta fold observed in many RNA binding proteins may further our understanding of the critical ZipA-FtsZ interaction.     

Solution structure of ribosomal protein L40E, a unique C4 zinc finger protein encoded by archaeon Sulfolobus solfataricus

The ribosomal protein L40E from archaeon Sulfolobus solfataricus is a component of the 50S ribosomal subunit. L40E is a 56-residue, highly basic protein that contains a C4 zinc finger motif, CRKC_X(10)_CRRC. Homologs are found in both archaea and eukaryotes but are not present in bacteria. Eukaryotic genomes encode L40E as a ubiquitin-fusion protein. L40E was absent from the crystal structure of euryarchaeota 50S ribosomal subunit. Here we report the three-dimensional solution structure of L40E by NMR spectroscopy. The structure of L40E is a three-stranded beta-sheet with a simple beta2beta1beta3 topology. There are two unique characteristics revealed by the structure. First, a large and ordered beta2-beta3 loop twists to pack across the one side of the protein. L40E contains a buried polar cluster comprising Lys19, Lys20, Cys22, Asn29, and Cys36. Second, the surface of L40E is almost entirely positively charged. Ten conserved basic residues are positioned on the two sides of the surface. It is likely that binding of zinc is essential in stabilizing the tertiary structure of L40E to act as a scaffold to create a broad positively charged surface for RNA and/or protein recognition.     

NMR structure of Streptomyces killer toxin-like protein, SKLP: further evidence for the wide distribution of single-domain betagamma-crystallin superfamily proteins

A protein isolated from the culture supernatant of the soil bacterium, Streptomyces sp. F-287, exhibits cytocidal effects for both budding and fission yeasts, and causes morphological changes of yeasts and filamentous fungi. This protein, which was the first killer toxin-like protein for yeasts identified in the Streptomyces microorganism, was named SKLP (Streptomyces killer toxin-like protein). Since the amino acid sequence of the protein, as determined by sequential Edman degradations, seemed to be unique, we determined the structure by NMR spectroscopy. Although the actual target of SKLP in yeasts has not been determined yet, the structure might give us a clue to characterize the targets. The solution structure of SKLP determined by NMR, however, turned out to be a single-domain crystallin-like protein, with two Greek key motifs and a short extra beta-strand at the N terminus. The final ensemble of 20 NMR structures overlaid onto their mean coordinate with rmsd values of 0.32(+/-0.06) A for the backbone atoms involved in the secondary structure elements. As a yeast killer toxin, WmKT, isolated from the yeast strain Williopsis mrakii also has a Greek key beta-barrel fold, we have made a detailed comparison of the structural features of SKLP with the other crystallin superfamily proteins. It is very interesting that SKLP has a unique electrostatic potential distribution on the molecular surface. Namely, one surface of the beta-barrel fold in SKLP has a large negatively charged region, with an isolated positive charge of the Arg62 side-chain at the center. The edge of this surface is surrounded by positively charged residues, including Arg31, Arg65 and Arg74. The salient features of the charge distribution on this surface and the cluster of Arg residues might be related to the target binding of SKLP.     

HU-alpha binds to the putative double-stranded DNA mimic HI1450 from Haemophilus influenzae

Recently, the solution structure of the hypothetical protein HI1450 from Haemophilus influenzae was solved as part of a structure-based effort to understand function. The distribution of its many negatively charged residues and weak structure and sequence homology to uracil DNA glycosylase inhibitor (Ugi) suggested that HI1450 may act as a double-stranded DNA (dsDNA) mimic. We present supporting evidence here and show that HI1450 interacts with the dsDNA-binding protein HU-alpha. The interaction between HI1450 and HU-alpha from H. influenzae is characterized using calorimetry and NMR spectroscopy. HU-alpha binds to HI1450 with a K(d) of 3.0 +/- 0.2 microM, which is similar in affinity to its interaction with dsDNA. Chemical shift perturbation data indicate that the beta1-strand of HI1450 and neighboring regions are most directly involved in interactions with HU-alpha. These results show that HI1450 and its structural homolog, Ugi, use similar parts of their structures to recognize DNA-binding proteins.     

NMR solution structure of plastocyanin from the photosynthetic prokaryote, Prochlorothrix hollandica

The solution structure of a divergent plastocyanin (PC) from the photosynthetic prokaryote Prochlorothrix hollandica was determined by homonuclear 1H NMR spectroscopy. Nineteen structures were calculated from 1222 distance restraints, yielding a family of structures having an average rmsd of 0.42 +/- 0.08 A for backbone atoms and 0.71 +/- 0.07 A for heavy atoms to the mean structure. No distance constraint was violated by more than 0.26 A in the structure family. Despite the low number of conserved residues shared with other PC homologues, the overall folding pattern of P. hollandica PC is similar to other PCs, in that the protein forms a two-sheet beta-barrel tertiary structure. The greatest variability among the backbone structures is seen in the loop region from residues 47-60. The differences seen in the P. hollandica PC homologue likely arise due to a small deletion of 2-4 residues compared to the PC consensus; this yields a less extended loop containing a short alpha-helix from residues Ala52-Leu55. Additionally, the protein has an altered hydrophobic patch thought to be important in binding reaction partners. Whereas the backbone structure is very similar within the loops of the hydrophobic region, the presence of two unique residues (Tyr12 and Pro14) yields a structurally different hydrophobic surface likely important in binding P. hollandica Photosystem I.     

The influence of stereospecific assignments on the determination of three-dimensional structures of proteins by nuclear magnetic resonance spectroscopy. Application to the sea anemone protein BDS-I

The influence of the stereospecific assignments of beta-methylene protons and the classification of chi 1 torsion angles on the definition of the three-dimensional structures of proteins determined from NMR data is investigated using the sea anemone protein BDS-I (43 residues) as a model system. Two sets of structures are computed. The first set comprises 42 converged structures (denoted STEREO structures) calculated on the basis of the complete list of restraints derived from the NMR data, consisting of 489 interproton and 24 hydrogen bonding distance restraints, supplemented by 23 phi backbone and 21 chi 1 side chain torsion angle restraints. The second set comprises 31 converged structures (denoted NOSTEREO structures) calculated from a reduced data set in which those restraints arising from stereospecific assignments, and the corresponding chi 1 torsion angle restraints, are explicitly omitted. The results show that the inclusion of the stereospecific restraints leads to a significant improvement in the definition of the structure of BDS-I, both with respect to the backbone and the detailed arrangement of the side chains. Average atomic rms differences between the individual structures and the mean structures for the backbone atoms are 0.67 +/- 0.12 A and 0.93 +/- 0.16 A for the STEREO and NOSTEREO structures, respectively; the corresponding values for all atoms are 0.90 +/- 0.17 A and 1.17 +/- 0.17 A, respectively. In addition, while the overall fold remains unchanged, there is a small but significant atomic displacement between the two sets of structures.     

A proton-NMR investigation of the fully reduced cytochrome c7 from Desulfuromonas acetoxidans. Comparison between the reduced and the oxidized forms.

The solution structure via 1H NMR of the fully reduced form of cytochrome c7 has been obtained. The protein sample was kept reduced by addition of catalytic amounts of Desulfovibrio gigas iron hydrogenase in H2 atmosphere after it had been checked that the presence of the hydrogenase did not affect the NMR spectrum. A final family of 35 conformers with rmsd values with respect to the mean structure of 8.7 +/- 1.5 nm and 12.4 +/- 1.3 nm for the backbone and heavy atoms, respectively, was obtained. A highly disordered loop involving residues 54-61 is present. If this loop is ignored, the rmsd values are 6.2 +/- 1.1 nm and 10.2 +/- 1.0 nm for the backbone and heavy atoms, respectively, which represent a reasonable resolution. The structure was analyzed and compared with the already available structure of the fully oxidized protein. Within the indetermination of the two solution structures, the result for the two redox forms is quite similar, confirming the special structural features of the three-heme cluster. A useful comparison can be made with the available crystal structures of cytochromes c3, which appear to be highly homologous except for the presence of a further heme. Finally, an analysis of the factors affecting the reduction potentials of the heme irons was performed, revealing the importance of net charges in differentiating the reduction potential when the other parameters are kept constant.     

Solution structure of ribosomal protein S28E from Methanobacterium thermoautotrophicum

The ribosomal protein S28E from the archaeon Methanobacterium thermoautotrophicum is a component of the 30S ribosomal subunit. Sequence homologs of S28E are found only in archaea and eukaryotes. Here we report the three-dimensional solution structure of S28E by NMR spectroscopy. S28E contains a globular region and a long C-terminal tail protruding from the core. The globular region consists of four antiparallel beta-strands that are arranged in a Greek-key topology. Unique features of S28E include an extended loop L2-3 that folds back onto the protein and a 12-residue charged C-terminal tail with no regular secondary structure and greater flexibility relative to the rest of the protein. The structural and surface resemblance to OB-fold family of proteins and the presence of highly conserved basic residues suggest that S28E may bind to RNA. A broad positively charged surface extending over one side of the beta-barrel and into the flexible C terminus may present a putative binding site for RNA.     

Tertiary structure and carbohydrate recognition by the chitin-binding domain of a hyperthermophilic chitinase from Pyrococcus furiosus

A chitinase is a hyperthermophilic glycosidase that effectively hydrolyzes both α and β crystalline chitins; that studied here was engineered from the genes PF1233 and PF1234 of Pyrococcus furiosus. This chitinase has unique structural features and contains two catalytic domains (AD1 and AD2) and two chitin-binding domains (ChBDs; ChBD1 and ChBD2). A partial enzyme carrying AD2 and ChBD2 also effectively hydrolyzes crystalline chitin. We determined the NMR and crystal structures of ChBD2, which significantly enhances the activity of the catalytic domain. There was no significant difference between the NMR and crystal structures. The overall structure of ChBD2, which consists of two four-stranded β-sheets, was composed of a typical β-sandwich architecture and was similar to that of other carbohydrate-binding module 2 family proteins, despite low sequence similarity. The chitin-binding surface identified by NMR was flat and contained a strip of three solvent-exposed Trp residues (Trp274, Trp308 and Trp326) flanked by acidic residues (Glu279 and Asp281). These acidic residues form a negatively charged patch and are a characteristic feature of ChBD2. Mutagenesis analysis indicated that hydrophobic interaction was dominant for the recognition of crystalline chitin and that the acidic residues were responsible for a higher substrate specificity of ChBD2 for chitin compared with that of cellulose. These results provide the first structure of a hyperthermostable ChBD and yield new insight into the mechanism of protein-carbohydrate recognition. This is important in the development of technology for the exploitation of biomass.     

Studies of a serum albumin-liposome complex as a model lipoprotein membrane.

Electron microscopy shows that the lipoprotein dispersions formed from the interaction of negatively charged liposomes with bovine serum albumin contain closed, vesicu lar, multilamellar structures. Discontinuous density gradient studies indicate that the lipoprotein suspensions are vesicles in which bovine serum albumin homogenously associates with lipid. Low angle X-ray diffraction results show that all the systems, positively and negatively charged, with and without protein, have the characteristic lamellar structure observed in biological membranes. The lamellar spacing (bilayer plus water layer) of negatively charged liposomes without bovine serum albumin is 55 A. The same lamellar separation in the positively charged system is 108 A. The lamellar spacing corresponding to bilayer, water, and protein for the negatively charged lipoprotein system is 93 A while that for the positively charged lipoprotein system is 91 A. These dimensions suggest that a layer of protein one molecule thick is incorporated between the lamellae bound to the surface of the bilayer. Wide angle X-ray diffraction results indicate no major effect of the protein on the 4.1 A spacing, characteristic of hexagonal packing of the hydrocarbon chains. A classical light scattering technique is used to show that the lipoprotein systems are osmotically active. The solute permeability exhibited by these lipoprotein systems follows the sequence (glucose smaller than arabinose smaller than malonamide smaller than glycerol). K+ diffusion from negatively charged lipoprotein systems is greater than that found for positively charged lipoprotein systems.     

Solution structure and backbone dynamics of the holo form of the frenolicin acyl carrier protein

During polyketide biosynthesis, acyl carrier proteins (ACPs) perform the central role of transferring polyketide intermediates between active sites of polyketide synthase. The 4'-phosphopantetheine prosthetic group of a holo-ACP is a long and flexible arm that can reach into different active sites and provide a terminal sulfhydryl group for the attachment of acyl groups through a thioester linkage. We have determined the solution structure and characterized backbone dynamics of the holo form of the frenolicin acyl carrier protein (fren holo-ACP) by nuclear magnetic resonance (NMR). Unambiguous assignments were made for 433 hydrogen atoms, 333 carbon atoms, and 84 nitrogen atoms, representing a total of 94.6% of the assignable atoms in this protein. From 879 meaningful NOEs and 45 angle constraints, a family of 24 structures has been calculated. The solution structure is composed of three major alpha-helices packed in a bundle with three additional short helices in intervening loops; one of the short helices slowly exchanges between two conformations. Superposition of the major helical regions on the mean structure yields average atomic rmsd values of 0.49 +/- 0.09 and 0.91 +/- 0.08 A for backbone and non-hydrogen atoms, respectively. Although the three-helix bundle fold is conserved among acyl carrier proteins involved in fatty acid synthases and polyketide synthases, a detailed comparison revealed that ACPs from polyketide biosynthetic pathways are more related to each other in tertiary fold than to their homologues from fatty acid biosynthetic pathways. Comparison of the free form of ACPs (NMR structures of fren ACP and the Bacillus subtilis ACP) with the substrate-bound form of ACP (crystal structure of butyryl-ACP from Escherichia coli) suggests that conformational exchange plays a role in substrate binding.     

Cold-adaptation in sea-water-borne signal proteins: sequence and NMR structure of the pheromone En-6 from the Antarctic ciliate Euplotes nobilii

Ciliates of Euplotes species constitutively secrete pleiotropic protein pheromones, which are capable to function as prototypic autocrine growth factors as well as paracrine inducers of mating processes. This paper reports the amino acid sequence and the NMR structure of the pheromone En-6 isolated from the antarctic species Euplotes nobilii. The 63-residue En-6 polypeptide chain forms three alpha-helices in positions 18-25, 36-40 and 46-56, which are arranged in an up-down-up three-helix bundle forming the edges of a distorted trigonal pyramid. The base of the pyramid is covered by the N-terminal heptadecapeptide segment, which includes a 3(10)-turn of residues 3-6. This topology is covalently anchored by four long-range disulfide bonds. Comparison with the smaller pheromones of E. raikovi, a closely related species living in temperate waters, shows that the two-pheromone families have the same three-helix bundle architecture. It then appears that cold-adaptation of the En proteins is primarily related to increased lengths of the chain-terminal peptide segments and the surface-exposed loops connecting the regular secondary structures, and to the presence of solvent-exposed clusters of negatively charged side-chains.     

Solution structure of the PDZ2 domain from human phosphatase hPTP1E and its interactions with C-terminal peptides from the Fas receptor

The solution structure of the second PDZ domain (PDZ2) from human phosphatase hPTP1E has been determined using 2D and 3D heteronuclear NMR experiments. The binding of peptides derived from the C-terminus of the Fas receptor to PDZ2 was studied via changes in backbone peptide and protein resonances. The structure is based on a total of 1387 nonredundant experimental NMR restraints including 1261 interproton distance restraints, 45 backbone hydrogen bonds, and 81 torsion angle restraints. Analysis of 30 lowest-energy structures resulted in rmsd values of 0.41 +/- 0.09 A for backbone atoms (N, Calpha, C') and 1.08 +/- 0.10 A for all heavy atoms, excluding the disordered N- and C-termini. The hPTP1E PDZ2 structure is similar to known PDZ domain structures but contains two unique structural features. In the peptide binding domain, the first glycine of the GLGF motif is replaced by a serine. This serine appears to replace a bound water observed in PDZ crystal structures that hydrogen bonds to the bound peptide's C-terminus. The hPTP1E PDZ2 structure also contains an unusually large loop following strand beta2 and proximal to the peptide binding site. This well-ordered loop folds back against the PDZ domain and contains several residues that undergo large amide chemical shift changes upon peptide binding. Direct observation of peptide resonances demonstrates that as many as six Fas peptide residues interact with the PDZ2 domain.     

Structure, dynamics, lipid binding, and physiological relevance of the putative GTPase-binding domain of Dictyostelium formin C

Dictyostelium Formin C (ForC) is involved in the regulation of local actin cytoskeleton reorganization (e.g. during cellular adhesion or migration). ForC contains formin homology 2 and 3 (FH2 and -3) domains and an N-terminal putative GTPase-binding domain (GBD) but lacks a canonical FH1 region. To better understand the role of the GBD, its structure, dynamics, lipid-binding properties, and cellular functions were analyzed by NMR and CD spectroscopy and by in vivo fluorescence microscopy. Moreover, the program CS-Rosetta was tested for the structure prediction based on chemical shift data only. The ForC GBD adopts an ubiquitin-like α/β-roll fold with an unusually long loop between β-strands 1 and 2. Based on the lipid-binding data, the presence of DPC micelles induces the formation of α-helical secondary structure and a rearrangement of the tertiary structure. Lipid-binding studies with a mutant protein and a peptide suggest that the β1-β2 loop is not relevant for these conformational changes. Whereas small amounts of negatively charged phosphoinositides (1,2-dioctanoyl-sn-glycero-3-(phosphoinositol 4,5-bisphosphate) and 1,2-dihexanoyl-sn-glycero-3-(phosphoinositol 3,4,5-trisphosphate)) lower the micelle concentration necessary to induce the observed spectral changes, other negatively charged phospholipids (1,2-dihexanoyl-sn-glycero-3-(phospho-L-serine) and 1,2-dihexanoyl-sn-glycero-3-phospho-(1'-rac-glycerol)) had no such effect. Interestingly, bicelles and micelles composed of diacylphosphocholines had no effect on the GBD structure. Our data suggest a model in which part of the large positively charged surface area of the GBD mediates localization to specific membrane patches, thereby regulating interactions with signaling proteins. Our cellular localization studies show that both the GBD and the FH3 domain are required for ForC targeting to cell-cell contacts and early phagocytic cups and macropinosomes.     

Determination of the three-dimensional solution structure of the antihypertensive and antiviral protein BDS-I from the sea anemone Anemonia sulcata: a study using nuclear magnetic resonance and hybrid distance geometry-dynamical simulated annealing

The three-dimensional solution structure of the antihypertensive and antiviral protein BDS-I from the sea anemone Anemonia sulcata has been determined on the basis of 489 interproton and 24 hydrogen-bonding distance restraints supplemented by 23 phi backbone and 21 chi 1 side-chain torsion angle restraints derived from nuclear magnetic resonance (NMR) measurements. A total of 42 structures is calculated by a hybrid metric matrix distance geometry-dynamical simulated annealing approach. Both the backbone and side-chain atom positions are well defined. The average atomic rms difference between the 42 individual SA structures and the mean structure obtained by averaging their coordinates is 0.67 +/- 0.12 A for the backbone atoms and 0.90 +/- 0.17 A for all atoms. The core of the protein is formed by a triple-stranded antiparallel beta-sheet composed of residues 14-16 (strand 1), 30-34 (strand 2), and 37-41 (strand 3) with an additional mini-antiparallel beta-sheet at the N-terminus (residues 6-9). The first and second strands of the triple-stranded antiparallel beta-sheet are connected by a long exposed loop (residues 17-30). A number of side-chain interactions are discussed in light of the structure.     

Solution structure of the Reps1 EH domain and characterization of its binding to NPF target sequences

The recently described EH domain recognizes proteins containing Asn-Pro-Phe (NPF) sequences. Using nuclear magnetic resonance (NMR) data, we determined the solution structure of the EH domain from the Reps1 protein and characterized its binding to linear and cyclic peptides derived from a novel targeting protein. The structure calculation included 1143 distance restraints and 122 angle restraints and resulted in structures with a root-mean-square deviation of 0.40 +/- 0.05 A for backbone atoms of superimposed secondary structural elements. The structure comprises two helix-loop-helix motifs characteristic of EF-hand domains. Titration data with NPF-containing peptides showed evidence of intermediate exchange on the NMR chemical shift time scale, which required an analysis that includes curve fitting to obtain accurate equilibrium constants and dissociation rate constants. The cyclic and linear peptides bound with similar affinities (Kd = 65 +/- 17 and 46 +/- 14 microM, respectively) and to the same hydrophobic pocket formed between helices B and C. The cyclic peptide formed a complex that dissociated more slowly (k(off) = 440 +/- 110 s(-1)) than the linear peptide (k(off) = 1800 +/- 250 s(-1)), but had little change in affinity because of the slower rate of association of the cyclic peptide. In addition, we characterized binding to a peptide containing a DPF sequence (Kd = 0.5 +/- 0.2 mM). The characterization of binding between the Reps1 EH domain and its target proteins provides information about their role in endocytosis.     

S4 charges move close to residues in the pore domain during activation in a K channel.

Voltage-gated ion channels respond to changes in the transmembrane voltage by opening or closing their ion conducting pore. The positively charged fourth transmembrane segment (S4) has been identified as the main voltage sensor, but the mechanisms of coupling between the voltage sensor and the gates are still unknown. Obtaining information about the location and the exact motion of S4 is an important step toward an understanding of these coupling mechanisms. In previous studies we have shown that the extracellular end of S4 is located close to segment 5 (S5). The purpose of the present study is to estimate the location of S4 charges in both resting and activated states. We measured the modification rates by differently charged methanethiosulfonate regents of two residues in the extracellular end of S5 in the Shaker K channel (418C and 419C). When S4 moves to its activated state, the modification rate by the negatively charged sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES(-)) increases significantly more than the modification rate by the positively charged [2-(trimethylammonium)ethyl] methanethiosulfonate, bromide (MTSET(+)). This indicates that the positive S4 charges are moving close to 418C and 419C in S5 during activation. Neutralization of the most external charge of S4 (R362), shows that R362 in its activated state electrostatically affects the environment at 418C by 19 mV. In contrast, R362 in its resting state has no effect on 418C. This suggests that, during activation of the channel, R362 moves from a position far away (>20 A) to a position close (8 A) to 418C. Despite its close approach to E418, a residue shown to be important in slow inactivation, R362 has no effect on slow inactivation or the recovery from slow inactivation. This refutes previous models for slow inactivation with an electrostatic S4-to-gate coupling. Instead, we propose a model with an allosteric mechanism for the S4-to-gate coupling.