S1 Binding Research Articles

Abstract 1464: Cardiac Myosin Binding Protein-C (cMyBP-C) Directly Modulates Actomyosin Binding and Kinetics Independent of LMM and S2 Binding.

BACKGROUND: While mutations in cMyBP-C constitute a common cause of FHC, its role in sarcomere contraction remains unclear. cMyBP-C binds to actin, titin and the S2 and LMM proteolytic domains of myosin. Through its numerous binding interactions cMyBP-C may act as a tether, restricting myosin and/or actomyosin function. We directly tested this hypothesis in the in vitro motility assay using either whole myosin or the myosin subfragments, HMM and S1 (which lack LMM and LMM-S2, respectively). METHODS AND RESULTS: The motility assay is an in vitro model of muscle contraction in which thin filaments are propelled across a myosin coated surface. The addition of cMyBP-C to the motility assay resulted in a concentration dependent reduction in actin filament velocity when using either whole myosin, HMM or S1, demonstrating that cMyBP-C inhibits thin filament velocity independent of LMM or S2 binding. Using whole myosin and thin filaments reconstituted with troponin/tropomyosin, the addition of cMyBP-C resulted in a 29% reduction in maximal velocity (P=0.002) with no effect on maximal force. At sub-maximal calcium, the pCa50 for velocity was increased (6.64 ± 0.06 vs. control, 6.44 ± 0.03, P=0.003) whereas the pCa50 for force was decreased (6.25 ± 0.09 vs. control, 6.55 ± 0.02, P=0.008). Thin filament activation by myosin strong-binding demonstrated an increased amount of myosin required to half maximally activate the thin filament in the presence of cMyBP-C, indicating that myosin binding to the thin filament is reduced with cMyBP-C. These findings were supported by co-sedimentation experiments which demonstrate that cMyBP-C competes with S1 for actin binding in the presence of ATP, with no effect on S1/actin binding in the absence of ATP. Finally, while the number of cross-bridges interacting with the thin filament is rate limiting for velocity at shorter filament lengths, this was not observed at longer filament lengths indicating that cMyBP- C directly modulates the kinetics of actomyosin. CONCLUSIONS: The effects of cMyBP-C on velocity and force demonstrate that cMyBP-C does not simply act as a tether but likely affects both the kinetics and the recruitment of myosin cross-bridges through its direct interaction with the myosin head and/or the actin filament.

D-Phenylglycinol-derived non-covalent factor Xa inhibitors: Effect of non-peptidic S4 linkage elements on affinity and anticoagulant activity

Analogs to a series of d-phenylglycinamide-derived factor Xa inhibitors were discovered. It was found that the S4 amide linkage can be replaced with an ether linkage to reduce the peptide character of the molecules and that this substitution leads to an increase in binding affinity that is not predicted based on modeling. Inhibitors which incorporate ether, amino, or alkyl S4 linkage motifs exhibit similar levels of binding affinity and also demonstrate potent in vitro functional activity, however, binding affinity in this series is strongly dependent on the nature of the S1 binding element.

The Regulation of Myosin Binding to Actin Filaments by Lethocerus Troponin

Lethocerus indirect flight muscle has two isoforms of troponin C, TnC-F1 and F2, which are unusual in having only a single C-terminal calcium binding site (site IV, isoform F1) or one C-terminal and one N-terminal site (sites IV and II, isoform F2). We show here that thin filaments assembled from rabbit actin and Lethocerus tropomyosin (Tm) and troponin (Tn) regulate the binding of rabbit myosin to rabbit actin in much the same way as the mammalian regulatory proteins. The removal of calcium reduces the rate constant for S1 binding to regulated actin about threefold, independent of which TmTn is used. This is consistent with calcium removal causing the TmTn to occupy the B or blocked state to about 70% of the total. The mid point pCa for the switch differed for TnC-F1 and F2 (pCa 6.9 and 6.0, respectively) consistent with the reported calcium affinities for the two TnCs. Equilibrium titration of S1 binding to regulated actin filaments confirms calcium regulated binding of S1 to actin and shows that in the absence of calcium the three actin filaments (TnC-F1, TnC-F2 and mammalian control) are almost indistinguishable in terms of occupancy of the B and C states of the filament. In the presence of calcium TnC-F2 is very similar to the control with ∼80% of the filament in the C-state and 10–15% in the fully on M-State while TnC-F1 has almost 50% in each of the C and M states. This higher occupancy of the M-state for TnC-F1, which occurs above pCa 6.9, is consistent with this isoform being involved in the calcium activation of stretch activation. However, it leaves unanswered how a C-terminal calcium binding site of TnC can activate the thin filament.

Prodrug-based design, synthesis, and biological evaluation of N-benzenesulfonylpiperidine derivatives as novel, orally active factor Xa inhibitors

We describe here our investigation of a new series of orally active fXa inhibitors based on a prodrug strategy. Solid-phase parallel synthesis identified a unique series of fXa inhibitors with a substituted benzenesulfonyl group as a novel S4 binding element. This series resulted in compound 39, which exhibited potent inhibitory activity against fXa (IC 50 = 13 nM) and excellent selectivity over thrombin (>7000-fold). The masking of its highly hydrophilic groups led to the creation of related prodrug 28, which demonstrated an anticoagulant effect after oral dosing.

N- and C-terminal residues of eIF1A have opposing effects on the fidelity of start codon selection

Translation initiation factor eIF1A stimulates preinitiation complex (PIC) assembly and scanning, but the molecular mechanisms of its functions are not understood. We show that the F131A,F133A mutation in the C-terminal tail (CTT) of eIF1A impairs recruitment of the eIF2-GTP-Met-tRNA(i)(Met) ternary complex to 40S subunits, eliminating functional coupling with eIF1. Mutating residues 17-21 in the N-terminal tail (NTT) of eIF1A also reduces PIC assembly, but in a manner rescued by eIF1. Interestingly, the 131,133 CTT mutation enhances initiation at UUG codons (Sui(-) phenotype) and decreases leaky scanning at AUG, while the NTT mutation 17-21 suppresses the Sui(-) phenotypes of eIF5 and eIF2beta mutations and increases leaky scanning. These findings and the opposite effects of the mutations on eIF1A binding to reconstituted PICs suggest that the NTT mutations promote an open, scanning-conducive conformation of the PIC, whereas the CTT mutations 131,133 have the reverse effect. We conclude that tight binding of eIF1A to the PIC is an important determinant of AUG selection and is modulated in opposite directions by residues in the NTT and CTT of eIF1A.

Activation Mechanism and Steady State Kinetics of Bruton's Tyrosine Kinase

Bruton's tyrosine kinase (BTK) is a member of the Tec non-receptor tyrosine kinase family that is involved in regulating B cell proliferation. To better understand the enzymatic mechanism of the Tec family of kinases, the kinetics of BTK substrate phosphorylation were characterized using a radioactive enzyme assay. We first examined whether autophosphorylation regulates BTK activity. Western blotting with a phosphospecific antibody revealed that BTK rapidly autophosphorylates at Tyr(551) within its activation loop in vitro. Examination of a Y551F BTK mutant indicated that phosphorylation of Tyr(551) causes a 10-fold increase in BTK activity. We then proceeded to characterize the steady state kinetic mechanism of BTK. Varying the concentrations of ATP and S1 peptide (biotin-Aca-AAAEEIY-GEI-NH2) revealed that BTK employs a ternary complex mechanism with KmATP = 84 +/- 20 microM and KmS1 = 37 +/- 8 microM. Inhibition studies were also performed to examine the order of substrate binding. The inhibitors ADP and staurosporine were both found to be competitive with ATP and non-competitive with S1, indicating binding of ATP and S1 to BTK is either random or ordered with ATP binding first. Negative cooperativity was also found between the S1 and ATP binding sites. Unlike ATP site inhibitors, substrate analog inhibitors did not inhibit BTK at concentrations less than 1 mm, suggesting that BTK may employ a "substrate clamping" type of kinetic mechanism whereby the substrate Kd is weaker than Km. This investigation of BTK provides the first detailed kinetic characterization of a Tec family kinase.

The role of the S1 binding site of carboxypeptidase M in substrate specificity and turn-over

The influence of the P1 amino acid on the substrate selectivity, the catalytic parameters K m and k cat, of carboxypeptidase M (CPM) (E.C. 3.4.17.12) was systematically studied using a series of benzoyl-Xaa–Arg substrates. CPM had the highest catalytic efficiency ( k cat/ K m) for substrates with Met, Ala and aromatic amino acids in the penultimate position and the lowest with amino acids with branched side-chains. Substrates with Pro in P1 were not cleaved in similar conditions. The P1 substrate preference of CPM differed from that of two other members of the carboxypeptidase family, CPN (CPN/CPE subfamily) and CPB (CPA/CPB subfamily). Aromatic P1 residues discriminated most between CPM and CPN. The type of P2 residue also influenced the k cat and K m of CPM. Extending the substrate up to P7 had little effect on the catalytic parameters. The substrates were modelled in the active site of CPM. The results indicate that P1-S1 interactions play a role in substrate binding and turn-over.

New Insights in the Role of Cardiac Myosin Binding Protein C As a Regulator of Cardiac Contractility

See related article, pages 884–890 Myosin Binding Protein C is a large multidomain protein that is found in the A-band region of the sarcomere where it associates with the thick filament.1 Mutations in cardiac myosin binding protein C (cMyBP-C) are a major cause of familial hypertrophic cardiomyopathy2 but the functions of cMyBP-C are not well resolved. In this issue Moss and colleagues (Stelzer et al3) provide important new insights in the role of cMyBP-C in the regulation of cardiac muscle contraction. A binding site for the light meromyosin (LMM) domain of myosin is found near the C-terminus of MyBP-C and a second site that binds the S2 domain of myosin is present near the N-terminus of MyBP-C.4 The cardiac MyBP-C isoform contains protein kinase A (PKA) phosphorylation sites within the S2 binding site that are absent in skeletal muscle isoforms. Although phosphorylating these residues abolishes binding to myosin S2,4 the functional role of this phosphorylation is not well understood. Stelzer et al3 examined the effect of PKA on stretch activation in skinned cardiac myocytes of wildtype and cMyBP-C KO mice. Their findings provide an important piece of the puzzle—cMyBP-C slows crossbridge cycling kinetics (confirming earlier results5) and, importantly, this effect can be relieved by PKA-based phosphorylation of cMyBP-C. It is well known from work initiated by Professor Pringle mid last century that stretch activation is most pronounced in asynchronous flight muscle, where it gives rise to wing beat frequencies that far exceed the capacity of the sarcoplasmic reticulum to activate and relax myofilaments.6 Although cardiac muscle largely relies on calcium cycling for activation and relaxation, several decades ago Steiger and colleagues7 and more recently Moss and colleagues8 have shown that stretch activation …

Mapping contacts of the S12–S7 intercistronic region of str operon mRNA with ribosomal protein S7 of E. coli

In E. coli, S7 initiates 30S ribosome assembly by binding to 16S rRNA. It also regulates translation of the S12 and S7 cistrons of the ‘streptomycin’ operon transcript by binding to the S12–S7 intercistronic region. Here, we describe the contacts of N-terminally His 6-tagged S7 with this region as mapped by UV-induced cross-linking. The cross-links are located at U(−34), U(−35), quite distant from the start codons of the two cistrons. In order to explain the mechanism of translational repression of S12–S7, we consider a possible conformational rearrangement of the intercistronic RNA structure induced by S7 binding.

Functional Homodimers and Heterodimers of Recombinant Smooth Muscle Tropomyosin

Skeletal and smooth muscle tropomyosin (Tm) require acetylation of their N-termini to bind strongly to actin. Tm containing an N-terminal alanine-serine (AS) extension to mimic acetylation has been widely used to increase binding. The current study investigates the ability of an N-terminal AS extension to mimic native acetylation for both alpha alpha and beta beta smooth Tm homodimers. We show that (1) AS alpha-Tm binds actin 100-fold tighter than alpha-Tm and 2-fold tighter than native smooth alphabeta-Tm, (2) beta-Tm requires an AS extension to bind actin, and (3) AS beta-Tm binds actin 10-fold weaker than AS alpha-Tm. Tm is present in smooth muscle tissues as >95% heterodimer; therefore, we studied the binding of recombinant alphabeta heterodimers with different AS extensions. This study shows that recombinant Tm requires an AS extension on both alpha and beta chains to bind like native Tm and that the alpha chain contributes more to actin binding than the beta chain. Once assembled onto an actin filament, all smooth muscle Tm's regulate S1 binding to actin Tm in the same way, irrespective of the presence of an AS extension.

Interactions of ribosomal protein S1 with DsrA and rpoS mRNA

Ribosomal protein S1 is shown to interact with the non-coding RNA DsrA and with rpoS mRNA. DsrA is a non-coding RNA that is important in controlling expression of the rpoS gene product in Escherichia coli. Photochemical crosslinking, quadrupole-time of flight tandem mass spectrometry, and peptide sequencing have identified an interaction between DsrA and S1 in the 30S ribosomal subunit. Purified S1 binds both DsrA ( K obs ∼6 × 10 6 M −1) and rpoS mRNA ( K obs ∼3 × 10 7 M −1). Ribonuclease probing experiments indicate that S1 binding has a weak but detectable effect on the secondary structure of DsrA or rpoS mRNA.

The Mechanism of Smooth Muscle Caldesmon-Tropomyosin Inhibition of the Elementary Steps of the Actomyosin ATPase

Caldesmon is a component of smooth muscle thin filaments that inhibits the actomyosin ATPase via its interaction with actin-tropomyosin. We have performed a comprehensive transient kinetic characterization of the actomyosin ATPase in the presence of smooth muscle caldesmon and tropomyosin. At physiological ratios of caldesmon to actin (1 caldesmon/7 actin monomers) actomyosin ATPase is inhibited by about 75%. Inhibitory caldesmon concentrations had little effect upon the rate of S1 binding to actin, actin-S1 dissociation by ATP, and dissociation of ADP from actin-S1 x ADP; however the rate of phosphate release from the actin-S1 x ADP x P(i) complex was decreased by more than 80%. In addition the transient of phosphate release displayed a lag of up to 200 ms. The presence of a lag phase indicates that a step on the pathway prior to phosphate release has become rate-limiting. Premixing the actin-tropomyosin filaments with myosin heads resulted in the disappearance of the lag phase. We conclude that caldesmon inhibition of the rate of phosphate release is caused by the thin filament being switched by caldesmon to an inactive state. The active and inactive states correspond to the open and closed states observed in skeletal muscle thin filaments with no evidence for the existence of a third, blocked state. Taken together these data suggest that at physiological concentrations, caldesmon controls the isomerization of the weak binding complex to the strong binding complex, and this causes the inhibition of the rate of phosphate release. This inhibition is sufficient to account for the inhibition of the steady state actomyosin ATPase by caldesmon and tropomyosin.

Interaction of the RNP1 Motif in PRT1 with HCR1 Promotes 40S Binding of Eukaryotic Initiation Factor 3 in Yeast

We found that mutating the RNP1 motif in the predicted RRM domain in yeast eukaryotic initiation factor 3 (eIF3) subunit b/PRT1 (prt1-rnp1) impairs its direct interactions in vitro with both eIF3a/TIF32 and eIF3j/HCR1. The rnp1 mutation in PRT1 confers temperature-sensitive translation initiation in vivo and reduces 40S-binding of eIF3 to native preinitiation complexes. Several findings indicate that the rnp1 lesion decreases recruitment of eIF3 to the 40S subunit by HCR1: (i) rnp1 strongly impairs the association of HCR1 with PRT1 without substantially disrupting the eIF3 complex; (ii) rnp1 impairs the 40S binding of eIF3 more so than the 40S binding of HCR1; (iii) overexpressing HCR1-R215I decreases the Ts(-) phenotype and increases 40S-bound eIF3 in rnp1 cells; (iv) the rnp1 Ts(-) phenotype is exacerbated by tif32-Delta6, which eliminates a binding determinant for HCR1 in TIF32; and (v) hcr1Delta impairs 40S binding of eIF3 in otherwise wild-type cells. Interestingly, rnp1 also reduces the levels of 40S-bound eIF5 and eIF1 and increases leaky scanning at the GCN4 uORF1. Thus, the PRT1 RNP1 motif coordinates the functions of HCR1 and TIF32 in 40S binding of eIF3 and is needed for optimal preinitiation complex assembly and AUG recognition in vivo.

Steady State Kinetic Mechanism of Bruton’s Tyrosine Kinase

Bruton’s Tyrosine Kinase (BTK) is a member of the Tec non-receptor tyrosine kinase family that is involved in regulating B cell proliferation. In order to better understand how both substrates and small molecule inhibitors interact with BTK, we studied the BTK steady-state kinetic mechanism. We first developed a bead-based, radioactive filtration assay to monitor BTK enzyme activity. We then used this assay to examine how BTK activity varied with the concentration of substrates ATP and S1 peptide (biotin-Aca-AAAEEIYGEI-NH2). These studies revealed that BTK employs a sequential mechanism with only modest communication between substrate binding sites (α = 2.4 ± 0.8). Maximum velocity was 121 ± 52 pmol/min/μ g, with Km(ATP) = 93 ± 13 μ M and Km (S1) = 45 ± 9 μ M. Dead-end inhibition studies were also performed to examine the order of substrate binding. The inhibitors ADP and staurosporine were both found to be competitive with ATP and non-competitive with S1, indicating binding of S1 to BTK is random. This kinetic characterization of BTK will aid in the identification of novel enzyme inhibitors that may be useful for treating rheumatoid arthritis or other inflammatory diseases.

Eukaryotic Translation Initiation Factor 3 (eIF3) and eIF2 Can Promote mRNA Binding to 40S Subunits Independently of eIF4G in Yeast

Recruitment of the eukaryotic translation initiation factor 2 (eIF2)-GTP-Met-tRNAiMet ternary complex to the 40S ribosome is stimulated by multiple initiation factors in vitro, including eIF3, eIF1, eIF5, and eIF1A. Recruitment of mRNA is thought to require the functions of eIF4F and eIF3, with the latter serving as an adaptor between the ribosome and the 4G subunit of eIF4F. To define the factor requirements for these reactions in vivo, we examined the effects of depleting eIF2, eIF3, eIF5, or eIF4G in Saccharomyces cerevisiae cells on binding of the ternary complex, other initiation factors, and RPL41A mRNA to native 43S and 48S preinitiation complexes. Depleting eIF2, eIF3, or eIF5 reduced 40S binding of all constituents of the multifactor complex (MFC), comprised of these three factors and eIF1, supporting a mechanism of coupled 40S binding by MFC components. 40S-bound mRNA strongly accumulated in eIF5-depleted cells, even though MFC binding to 40S subunits was reduced by eIF5 depletion. Hence, stimulation of the GTPase activity of the ternary complex, a prerequisite for 60S subunit joining in vitro, is likely the rate-limiting function of eIF5 in vivo. Depleting eIF2 or eIF3 impaired mRNA binding to free 40S subunits, but depleting eIF4G led unexpectedly to accumulation of mRNA on 40S subunits. Thus, it appears that eIF3 and eIF2 are more critically required than eIF4G for stable binding of at least some mRNAs to native preinitiation complexes and that eIF4G has a rate-limiting function at a step downstream of 48S complex assembly in vivo.

Maximal Activation of Skeletal Muscle Thin Filaments Requires Both Rigor Myosin S1 and Calcium

The regulation by calcium and rigor-bound myosin-S1 of the rate of acceleration of 2'-deoxy-3'-O-(N-methylanthraniloyl)ADP (mdADP) release from myosin-mdADP-P(i) by skeletal muscle thin filaments (reconstituted from actin-tropomyosin-troponin) was measured using double mixing stopped-flow fluorescence with the nucleotide substrate 2'-deoxy-3'-O-(N-methylanthraniloyl). The predominant mechanism of regulation is the acceleration of product dissociation by a factor of approximately 200 by thin filaments in the fully activated conformation (bound calcium and rigor S1) relative to the inhibited conformation (no bound calcium or rigor S1). In contrast, only 2-3-fold regulation is due to a change in actin affinity such as would be expected by "steric blocking" of the myosin binding site of the thin filament by tropomyosin. The binding of one ligand (either calcium or rigor-S1) produces partial activation of the rate of product dissociation, but the binding of both is required to maximally accelerate product dissociation to a rate similar to that obtained with F-actin in the absence of regulatory proteins. The data support an allosteric regulation model in which the binding of either calcium or rigor S1 alone to the thin filament shifts the equilibrium in favor of the active conformation, but full activation requires binding of both ligands.

The 1.8 Å crystal structure of a proteinase K‐like enzyme from a psychrotroph Serratia species

Proteins from organisms living in extreme conditions are of particular interest because of their potential for being templates for redesign of enzymes both in biotechnological and other industries. The crystal structure of a proteinase K-like enzyme from a psychrotroph Serratia species has been solved to 1.8 A. The structure has been compared with the structures of proteinase K from Tritirachium album Limber and Vibrio sp. PA44 in order to reveal structural explanations for differences in biophysical properties. The Serratia peptidase shares around 40 and 64% identity with the Tritirachium and Vibrio peptidases, respectively. The fold of the three enzymes is essentially identical, with minor exceptions in surface loops. One calcium binding site is found in the Serratia peptidase, in contrast to the Tritirachium and Vibrio peptidases which have two and three, respectively. A disulfide bridge close to the S2 site in the Serratia and Vibrio peptidases, an extensive hydrogen bond network in a tight loop close to the substrate binding site in the Serratia peptidase and different amino acid sequences in the S4 sites are expected to cause different substrate specificity in the three enzymes. The more negative surface potential of the Serratia peptidase, along with a disulfide bridge close to the S2 binding site of a substrate, is also expected to contribute to the overall lower binding affinity observed for the Serratia peptidase. Clear electron density for a tripeptide, probably a proteolysis product, was found in the S' sites of the substrate binding cleft.

Alteration of the Factor X Zymogen to Protease Transition Provides Evidence for Allosteric Linkage between the S1 and FVa Binding Sites.

The zymogen to protease transition in the chymotrypsin-like serine protease family follows a well described mechanism in which bond cleavage at a highly conserved site (Arg15-Ile16; chymotrypsin numbering system) results in the unmasking of a new N-terminus that acts as an intramolecular ligand for Asp194. This new salt-bridge drives a conformational change in the so-called “activation domain”, surface loops consisting of the S1 specificity pocket, oxyanion hole, autolysis loop, and sodium biding site. It is well documented in the trypsin system that Ile16-Asp194 internal salt-bridge formation is allosterically linked to the S1 specificity site; that is changes at one site influence the other and vice versa. Blood coagulation factor Xa (FXa) reversibly associates with its cofactor factor Va (FVa) on a membrane surface in the presence of Ca2+ ions with high affinity; an interaction which is not mimicked by the zymogen FX. To determine whether the FX zymogen to protease transition contributes to the expression of a high affinity FVa binding site, we constructed a series of FXa variants which are shifted along this transition pathway. To generate these “zymogen-like” proteins, we made several substitutions at position 16 or 17, with the intent of destabilizing the intramolecular salt bridge to varying degrees. Following a series of preliminary experiments, three mutants were chosen for expression, purification, and activation with RVV-X: I16L, I16G, and V17A. Kinetic studies using peptidyl substrates and active site directed probes revealed that I16L and V17A have an impaired ability to bind these probes (15 to 25-fold increase in the Km or Ki) while the rate of catalysis (kcat) was reduced by 3-fold compared to wild-type FXa (wtFXa; plasma-derived and recombinant). The I16G variant was not inhibited by any of the probes examined and its chromogenic activity was severely impaired (>500 to 1000-fold), precluding calculation of kinetic parameters. These data are consistent with the idea that destabilization of internal salt-bridge formation (Ile16-Asp194) influences binding at the S1 specificity site. In contrast to these results, assembly of I16L and V17A into prothrombinase almost completely restored the Km for peptidyl substrates while the kcat was still 3-fold reduced, indicating that FVa binding can rescue binding at the active site. Surprisingly, even the Km value for I16G was almost completely restored (3-fold increased compared to wtFXa) when assembled in prothrombinase; however a 60-fold reduction in the kcat was found. Consistent with these data, kinetic studies using prothrombin or prethrombin-1 revealed that each of the FXa variants had a normal Km value when assembled in prothrombinase; while the kcat values where reduced to a similar extent as for the chromogenic substrates. Overall our data indicate that direct binding of these FXa variants to FVa rescues binding at S1 site, suggesting allosteric linkage exists between these sites. Thus the FX zymogen to protease transition not only influences the formation of the S1 pocket, but also contributes in a substantial way to the formation of a FVa binding site.

The discovery of fluoropyridine-based inhibitors of the factor VIIa/TF complex—Part 2

The activated factor VII/tissue factor complex (FVIIa/TF) is known to play a key role in the formation of blood clots. Inhibition of this complex may lead to new antithrombotic drugs. A fluoropyridine-based series of FVIIa/TF inhibitors was discovered which utilized a diisopropylamino group for binding in the S2 and S3 binding pockets of the active site of the enzyme complex. In this series, an enhancement in binding affinity was observed by substitution at the 5-position of the hydroxybenzoic acid sidechain. An X-ray crystal structure indicates that amides at this position may increase inhibitor binding affinity through interactions with the S1′/S2′ pocket.

Fibroblast growth factor 3, a protein with a dual subcellular fate, is interacting with human ribosomal protein S2

The secreted isoform of fibroblast growth factor 3 (FGF3) induces a mitogenic cell response, while the nuclear form inhibits cell proliferation. Recently, we identified a nucleolar FGF3-binding protein which is implicated in processing of pre-rRNA as a possible target of nuclear FGF3 signalling. Here, we report a second candidate protein identified by a yeast two-hybrid screen for nuclear FGF3 action, ribosomal protein S2, rpS2. Recombinant rpS2 binds to in vitro translated FGF3 and to nuclear FGF3 extracted from transfected COS-1 cells. Characterization of the FGF3 binding domain of rpS2 showed that both the Arg-Gly-rich N-terminal region and a short carboxyl-terminal sequence of rpS2 are necessary for FGF3 binding. Mapping the S2 binding domains of FGF3 revealed that these domains are important for both NoBP and rpS2 interaction. Transient co-expression of rpS2 and nuclear FGF3 resulted in a reduced nucleolar localization of the FGF. These findings suggest that the nuclear form of FGF3 inhibits cell proliferation by interfering with ribosomal biogenesis.