Molecular interactions between ribosomal proteins — An analysis of S7-S9, S7-S19, S9-S19 and S7-S9-S19 interactions

Ribosomal proteins S7, S9 and S 19 fromEscherichia coli have been studied by the sedimentation equilibrium technique for possible intermolecular interaction between pairs of proteins as well as in a mixture of 3 proteins. The proteins were isolated to a purity greater than 95% and were characterized in the reconstitution buffer. It was observed that none of the proteins has a tendency to self-associate in the concentration range studied in the temperature range 3–6°C. Protein S9 behaves differently in the presence of other proteins. Analysis of the sedimentation equilibrium data for S7-S9, S9-S19 and S7-S9-S19 complexes revealed the need for considering the presence of a component of higher molecular weight in the system along with the monomers and their complexes to provide a meaningful curve-fitting of the data. Proteins S7 and S19 were found to interact with an equilibrium constant of association of 3 ± 2 × 10⁴ M⁻¹ at 3°C with a Gibbs free energy of interaction ΔG° of −5·7 kcal/mol. These data are useful for the consideration of the stabilization of the 3 0S subunit through protein-protein interactions and also help in building a topographical model of the proteins of the small subunit from an energetics point of view. Part of this work was carried out at the Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030, USA.     

Probing pockets S2-S4' of the gamma-secretase active site with (hydroxyethyl)urea peptidomimetics

(Hydroxyethyl)urea peptidomimetics are potent inhibitors of gamma-secretase that are accessible in a few synthetic steps. Systematic alteration of P2-P4' revealed that the corresponding S2-S4' active site pockets accommodate a variety of substituents, consistent with the fact that this protease cleaves a variety of single-pass membrane proteins; however, phenylalanine is not well tolerated at P2'. A compound spanning P2-P3' was identified as a low nM inhibitor of gamma-secretase activity both in cells and under cell-free conditions.     

Conformational similarity in the activation of caspase-3 and -7 revealed by the unliganded and inhibited structures of caspase-7

Caspase-mediated apoptosis has important roles in normal cell differentiation and aging and in many diseases including cancer, neuromuscular disorders and neurodegenerative diseases. Therefore, modulation of caspase activity and conformational states is of therapeutic importance. We report crystal structures of a new unliganded conformation of caspase-7 and the inhibited caspase-7 with the tetrapeptide Ac-YVAD-Cho. Different conformational states and mechanisms for substrate recognition have been proposed based on unliganded structures of the redundant apoptotic executioner caspase-3 and -7. The current study shows that the executioner caspase-3 and -7 have similar conformations for the unliganded active site as well as the inhibitor-bound active site. The new unliganded caspase-7 structure exhibits the tyrosine flipping mechanism in which the Tyr230 has rotated to block entry to the S2 binding site similar to the active site conformation of unliganded caspase-3. The inhibited structure of caspase-7/YVAD shows that the P4 Tyr binds the S4 region specific to polar residues at the expense of a main chain hydrogen bond between the P4 amide and carbonyl oxygen of caspase-7 Gln 276, which is similar to the caspase-3 complex. This new knowledge of the structures and conformational states of unliganded and inhibited caspases will be important for the design of drugs to modulate caspase activity and apoptosis.     

Critical intra-linker interactions of HCN1-encoded pacemaker channels revealed by interchange of S3-S4 determinants

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels contribute to the spontaneous rhythmic activities in cardiac and neuronal cells. Recently, we reported that the S3-S4 linker of HCN1 channels influences activation, and that part of the linker is helical with the determinants G231, M232, and E235 clustered on one side. Here we explored the undefined role of the G(231)E(235)M(232) triplet by systematic substitutions. Replacing G231 or M232 next to the "neighboring" E235 in the S3-S4 helix with an anionic residue (i.e., G231E, M232E) rendered channels non-functional although they were localized on the membrane surface. Interestingly, this loss of function could be readily rescued either by introducing a countercharge at position 235 (G231E/E235R, M232E/E235R) or by interchanging residues 231 or 232 and 235 (G231E/E235G, M232E/E235M). We conclude that residues 231, 232, and 235 are in close spatial proximity to each other, and uniquely interact with one another to shape the phenotypes of HCN channels.     

Revisiting the S2 specificity of papain by structural analogs of Phe

Papain characteristically has a strong preference for encoded L-aromatic amino acids (Phe > Tyr) at P2 position. We re-examined papain S2 specificity using structural analogs of Phe, in fluorogenic substrates of the series: dansyl-Xaa-Arg-Ala-Pro-Trp (Xaa = P2 residue). Kinetic analyses showed that the S2 pocket accommodates a broad spectrum of Phe derivatives. Papain is poorly stereoselective towards Dns-(D/L)-Phe-Arg-Ala-Pro-Trp and binding is not critically affected by replacement of the benzyl ring by the non-aromatic lateral chain of cyclohexylalanine. The Km was significantly improved by mono- and di-chlorination of Phe, or by its substitution by an electronegative group-like NO2, but the specificity constant was unchanged. Shortening or lengthening the side chain by adding or removing a methylene group impairs the P2/S2 interactions significantly, as do constrained structural analogs of Phe. Incorporation of benzyl-substituted phenylalanyl amino acid could help to design peptide-derived inhibitors with greater affinity and bioavailability.     

Engineering substrate preference in subtilisin: structural and kinetic analysis of a specificity mutant

Bacillus subtilisin has been a popular model protein for engineering altered substrate specificity. Although some studies have succeeded in increasing the specificity of subtilisin, they also demonstrate that high specificity is difficult to achieve solely by engineering selective substrate binding. In this paper, we analyze the structure and transient state kinetic behavior of Sbt160, a subtilisin engineered to strongly prefer substrates with phenylalanine or tyrosine at the P4 position. As in previous studies, we measure improvements in substrate affinity and overall specificity. Structural analysis of an inactive version of Sbt160 in complex with its cognate substrate reveals improved interactions at the S4 subsite with a P4 tyrosine. Comparison of transient state kinetic behavior against an optimal sequence (DFKAM) and a similar, but suboptimal, sequence (DVRAF) reveals the kinetic and thermodynamic basis for increased specificity, as well as the limitations of this approach. While highly selective substrate binding is achieved in Sbt160, several factors cause sequence specificity to fall short of that observed with natural processing subtilisins. First, for substrate sequences which are nearly optimal, the acylation reaction becomes faster than substrate dissociation. As a result, the level of discrimination among these substrates diminishes due to the coupling between substrate binding and the first chemical step (acylation). Second, although Sbt160 has 24-fold higher substrate affinity for the optimal substrate DFKAM than for DVRAF, the increased substrate binding energy is not translated into improved transition state stabilization of the acylation reaction. Finally, as interactions at subsites become stronger, the rate-determining step in peptide hydrolysis changes from acylation to product release. Thus, the release of the product becomes sluggish and leads to a low k(cat) for the reaction. This also leads to strong product inhibition of substrate turnover as the reaction progresses. The structural and kinetic analysis reveals that differences in the binding modes at subsites for substrates, transition states, and products are subtle and difficult to manipulate via straightforward protein engineering. These findings suggest several new strategies for engineering highly sequence selective enzymes.     

Thermodynamic and kinetic properties of amino-terminal and S4-S5 loop HERG channel mutants under steady-state conditions

Gating kinetics and underlying thermodynamic properties of human ether-a-go-go-related gene (HERG) K⁺ channels expressed in Xenopus oocytes were studied using protocols able to yield true steady-state kinetic parameters. Channel mutants lacking the initial 16 residues of the amino terminus before the conserved eag/PAS region showed significant positive shifts in activation voltage dependence associated with a reduction of z_g values and a less negative ΔG_o, indicating a deletion-induced displacement of the equilibrium toward the closed state. Conversely, a negative shift and an increased ΔG_o, indicative of closed-state destabilization, were observed in channels lacking the amino-terminal proximal domain. Furthermore, accelerated activation and deactivation kinetics were observed in these constructs when differences in driving force were considered, suggesting that the presence of distal and proximal amino-terminal segments contributes in wild-type channels to specific chemical interactions that raise the energy barrier for activation. Steady-state characteristics of some single point mutants in the intracellular loop linking S4 and S5 helices revealed a striking parallelism between the effects of these mutations and those of the amino-terminal modifications. Our data indicate that in addition to the recognized influence of the initial amino-terminus region on HERG deactivation, this cytoplasmic region also affects activation behavior. The data also suggest that not only a slow movement of the voltage sensor itself but also delaying its functional coupling to the activation gate by some cytoplasmic structures possibly acting on the S4-S5 loop may contribute to the atypically slow gating of HERG.     

Structural determinants for the action of grayanotoxin in D1 S4-S5 and D4 S4-S5 intracellular linkers of sodium channel alpha-subunits

We located a novel binding site for grayanotoxin on the cytoplasmic linkers of voltage-dependent cardiac (rH1) or skeletal-muscle (mu 1) Na(+) channel isoforms (segments S4-S5 in domains D1 and D4), using the alanine scanning substitution method. GTX-modification of Na(+) channels, transiently expressed in HEK 293 cells, was evaluated under whole-cell voltage clamp, from the ratio of maximum chord conductance for modified and unmodified Na(+) channels. In mu 1, mutations K237A, L243A, S246A, K248A, K249A, L250A, S251A, or T1463A, caused a moderate, but statistically significant decrease in this ratio. On making corresponding mutations in rH1, only L244A dramatically reduced the ratio. Because in mu 1, the serine at position 251 is the only heterologous residue with respect to rH1 (Ala-252), we made a double mutant L243A&S251A to match the sequence of mu 1 and rH1 in S4-S5 linkers of both domains. This double mutation resulted in a significant decrease in the ratio, to the same extent as L244A substitution in rH1 did, indicating that the site at Leu-244 in rH1 or at Leu-243 in mu 1 is a novel one, exhibiting a synergistic effect of grayanotoxin.     

A novel S100 target conformation is revealed by the solution structure of the Ca2+-S100B-TRTK-12 complex

The Alzheimer-linked neural protein S100B is a signaling molecule shown to control the assembly of intermediate filament proteins in a calcium-sensitive manner. Upon binding calcium, a conformational change occurs in S100B exposing a hydrophobic surface for target protein interactions. The synthetic peptide TRTK-12 (TRTKIDWNKILS), derived from random bacteriophage library screening, bears sequence similarity to several intermediate filament proteins and has the highest calcium-dependent affinity of any target molecule for S100B to date (K(d) <1 microm). In this work, the three-dimensional structure of the Ca(2+)-S100B-TRTK-12 complex has been determined by NMR spectroscopy. The structure reveals an extended, contiguous hydrophobic surface is formed on Ca(2+)-S100B for target interaction. The TRTK-12 peptide adopts a coiled structure that fits into a portion of this surface, anchored at Trp(7), and interacts with multiple hydrophobic contacts in helices III and IV of Ca(2+)-S100B. This interaction is strikingly different from the alpha-helical structures found for other S100 target peptides. By using the TRTK-12 interaction as a guide, in combination with other available S100 target structures, a recognition site on helix I is identified that may act in concert with the TRTK-12-binding site from helices III and IV. This would provide a larger, more complex site to interact with full-length target proteins and would account for the promiscuity observed for S100B target protein interactions.     

Helical secondary structure of the external S3-S4 linker of pacemaker (HCN) channels revealed by site-dependent perturbations of activation phenotype

If, encoded by the hyperpolarization-activated cyclic nucleotide-modulated channel family (HCN1-4), contributes significantly to neuronal and cardiac pacing. Recently, we reported that the S3-S4 residue Glu-235 of HCN1 influences activation by acting as a surface charge. However, it is uncertain whether other residues of the external S3-S4 linker are also involved in gating. Furthermore, the secondary conformation of the linker is not known. Here we probed the structural and functional role of the HCN1 S3-S4 linker by introducing systematic mutations into the entire linker (defined as 229-237) and studying their effects. We found that the mutations K230A (-62.2 +/- 3.4 mV versus -72.2 +/- 1.7 mV of wild type (WT)), G231A (-64.4 +/- 1.3 mV), M232A (V(1/2) = -63.1 +/- 1.1 mV), and E235G (-65.4 +/- 1.5 mV) produced depolarizing activation shifts. Although E229A and M232A decelerated gating kinetics (<13- and 3-fold, respectively), K230A and G231A accelerated both activation and deactivation (< approximately 2-3-fold). D233A, S234A, V236A, and Y237A channels exhibited WT properties (p > 0.05). Shortening the linker (EVY235-237deltadeltadelta) caused depolarizing activation shift and slowed kinetics that could not be explained by removing the charge at position 235 alone. Secondary structural predictions by the modeling algorithms SSpro2 and PROF, along with refinements by our experimental data, suggest that part of the S3-S4 linker conforms a helical structure with the functionally important residues Met-232, Glu-235, and Gly-231 (|deltadeltaG|>1 kcal/mol) clustered on one side.     

Functional multiplicity of motor molecules revealed by a simple kinetic analysis.

We present a simple analytical solution for a kinetic model of motor molecule function with multiple arms. This model has a rate of motion proportional to the probability that all arms in a complex are detached from the cytoskeleton and, therefore, we refer to it as obligate cooperativity. The model has the form: v = Vmax/(1 + q/S)n, where Vmax is the maximum velocity, the product nq is the effective Michaelis constant at high [ATP], and n is the number of arms. Values of n = 2 and n = 1 give good fits to the heavy meromyosin and myosin S1 sliding velocity data, respectively, consistent with the number of active sites. Despite the complexity of the eukaryotic axoneme, beat frequency data from Chlamydomonas wild-type and oda mutants are also fit by this model.     

The S4-S5 linker of KCNQ1 channels forms a structural scaffold with the S6 segment controlling gate closure

In vivo, KCNQ1 α-subunits associate with the β-subunit KCNE1 to generate the slowly activating cardiac potassium current (I(Ks)). Structurally, they share their topology with other Kv channels and consist out of six transmembrane helices (S1-S6) with the S1-S4 segments forming the voltage-sensing domain (VSD). The opening or closure of the intracellular channel gate, which localizes at the bottom of the S6 segment, is directly controlled by the movement of the VSD via an electromechanical coupling. In other Kv channels, this electromechanical coupling is realized by an interaction between the S4-S5 linker (S4S5(L)) and the C-terminal end of S6 (S6(T)). Previously we reported that substitutions for Leu(353) in S6(T) resulted in channels that failed to close completely. Closure could be incomplete because Leu(353) itself is the pore-occluding residue of the channel gate or because of a distorted electromechanical coupling. To resolve this and to address the role of S4S5(L) in KCNQ1 channel gating, we performed an alanine/tryptophan substitution scan of S4S5(L). The residues with a "high impact" on channel gating (when mutated) clustered on one side of the S4S5(L) α-helix. Hence, this side of S4S5(L) most likely contributes to the electromechanical coupling and finds its residue counterparts in S6(T). Accordingly, substitutions for Val(254) resulted in channels that were partially constitutively open and the ability to close completely was rescued by combination with substitutions for Leu(353) in S6(T). Double mutant cycle analysis supported this cross-talk indicating that both residues come in close contact and stabilize the closed state of the channel.     

A structural basis for S100 protein specificity derived from comparative analysis of apo and Ca(2+)-calcyclin

Calcyclin is a homodimeric protein belonging to the S100 subfamily of EF-hand Ca(2+)-binding proteins, which function in Ca(2+) signal transduction processes. A refined high-resolution solution structure of Ca(2+)-bound rabbit calcyclin has been determined by heteronuclear solution NMR. In order to understand the Ca(2+)-induced structural changes in S100 proteins, in-depth comparative structural analyses were used to compare the apo and Ca(2+)-bound states of calcyclin, the closely related S100B, and the prototypical Ca(2+)-sensor protein calmodulin. Upon Ca(2+) binding, the position and orientation of helix III in the second EF-hand is altered, whereas the rest of the protein, including the dimer interface, remains virtually unchanged. This Ca(2+)-induced structural change is much less drastic than the "opening" of the globular EF-hand domains that occurs in classical Ca(2+) sensors, such as calmodulin. Using homology models of calcyclin based on S100B, a binding site in calcyclin has been proposed for the N-terminal domain of annexin XI and the C-terminal domain of the neuronal calcyclin-binding protein. The structural basis for the specificity of S100 proteins is discussed in terms of the variation in sequence of critical contact residues in the common S100 target-binding site.     

Caspase-8 specificity probed at subsite S(4): crystal structure of the caspase-8-Z-DEVD-cho complex

Caspase-8 is an initiator enzyme in the Fas-mediated pathway of which the downstream executioner caspase-3 is a physiological target. Caspases are cysteine proteases that are specific for substrates with an aspartic acid residue at the P(1) position and have an optimal recognition motif that incorporates four amino acid residues N-terminal to the cleavage site. Caspase-8 has been classified as a group III caspase member because it shows a preference for a small hydrophobic residue at the P(4) substrate position. We report the X-ray crystallographic structure of caspase-8 in complex with benzyloxycarbonyl-Asp-Glu-Val-Asp-aldehyde (Z-DEVD), a specific group II caspase inhibitor. The structure shows that the inhibitor interacts favourably with the enzyme in subsite S(4). Kinetic data reveal that Z-DEVD (K(i) 2 nM) is an almost equally potent inhibitor of caspase-8 as the specific group III inhibitor Boc-IETD-aldehyde (K(i) 1 nM). In view of this finding, the original classification of caspases into three specificity groups needs to be modified, at least for caspase-8, which tolerates small hydrophobic residues as well as the acidic residue Asp in subsite S(4). We propose that the subsite S(3) must be considered as an important specificity-determining factor.     

Dissecting the structural and functional roles of the S3-S4 linker of pacemaker (hyperpolarization-activated cyclic nucleotide-modulated) channels by systematic length alterations

If or Ih, a key player in neuronal and cardiac pacing, is encoded by the hyperpolarization-activated cyclic nucleotide-modulated (HCN) channel gene family. We have recently reported that the S3-S4 linker (i.e. residues 229EKGMDSEVY237 of HCN1) prominently influences the activation phenotypes of HCN channels and that part of the linker may conform a secondary helical structure. Here we further dissected the structural and functional roles of this linker by systematic alterations of its length. In contrast to voltage-gated K+ channels, complete deletion of the S3-S4 linker (Delta229-237) did not produce functional channels. Similarly, the deletions Delta229-234, Delta232-234, and Delta232-237 also abolished normal current activity. Interestingly, Delta229-231, Delta233-237, Delta234-237, Delta235-237, Delta229-231/Delta233-237, Delta229-231/Delta234-237, and Delta229-231/Delta235-237 all yielded robust hyperpolarization-activated inward currents, indicating that loss-of-function caused by deletion could be rescued by keeping the single functionally important residue Met232 alone. Whereas shortening the linker by deletion generally shifted steady-state activation in the depolarizing direction (e.g. DeltaV1/2 of Delta229-231, Delta233-237, Delta235-237 > +10 mV relative to wild type), linker prolongation by duplicating the entire linker (Dup229-237) or by glutamine insertion (InsQ233Q, InsQQ233QQ and InsQQQ233QQQ, or Ins237QQQ) produced length-dependent progressive hyperpolarizing activation shifts (-35 mV < DeltaV1/2 < -4 mV). Based on these results, we conclude that only Met232 is prerequisite for channels to function, but the length and other constituents of the S3-S4 linker shape the ultimate activation phenotype. Our results also highlight several evolutionary similarities and differences between HCN and voltage-gated K+ channels. Manipulations of the S3-S4 linker length may provide a flexible approach to customize HCN gating for engineering electrically active cells (such as stem cell-derived neuronal and cardiac pacemakers) for gene- and cell-based therapies.     

Unexpected substrate specificity of T4 DNA ligase revealed by in vitro selection.

We have used in vitro selection techniques to characterize DNA sequences that are ligated efficiently by T4 DNA ligase. We find that the ensemble of selected sequences ligates about 50 times as efficiently as the random mixture of sequences used as the input for selection. Surprisingly many of the selected sequences failed to produce a match at or close to the ligation junction. None of the 20 selected oligomers that we sequenced produced a match two bases upstream from the ligation junction.