Effector Binding Sites Research Articles

Allosteric Networks in Thrombin

Serine proteases are found ubiquitously in both eukaryotes and prokaryotes however the dynamic motions of this largest peptidase family remains unknown. All serine proteases have a double β-barrel core surrounded by connecting loops and helices, but compared to the prototypical serine protease, chymotrypsin, thrombin has more extended loops that are thought to impart greater specificity. We analyzed apo-thrombin and active site-bound (PPACK-thrombin) using a combination of MD, accelerated MD and NMR. Community analysis of the MD ensembles identified groups of residues linked through correlated motions and physical contacts. AMD simulations, calibrated on measured residual dipolar couplings, revealed correlated loop motions connecting the active site with distal allosteric regulator binding sites. The backbone dynamics profile (from ps to ms motions) of thrombin was determined in both states using R1, R2, 15N-{1H}NOEs, TROSY Hahn-Echo, and relaxation dispersion experiments. The apo thrombin backbone is highly dynamic and shows motions across multiple time scales. The various surface loops move on different time scales. The substrate-binding loops showed intermediate time scale motions that are quenched upon PPACK binding. TROSY Hahn-echo and relaxation dispersion experiments reveal a large number of residues throughout the protein undergoing temporally correlated µs-ms motions. These include the Na+-binding loop and the β-strand connecting exosite 1 and the active site, both of which are implicated in allosteric coupling of effector binding sites with the active site. The results show a network of slowly exchanging residues extends through the entire apo-thrombin molecule.

McbR/YncC: implications for the mechanism of ligand and DNA binding by a bacterial GntR transcriptional regulator involved in biofilm formation.

MqsR-controlled colanic acid and biofilm regulator (McbR, also known as YncC) is the protein product of a highly induced gene in early Escherichia coli biofilm development and has been regarded as an attractive target for blocking biofilm formation. This protein acts as a repressor for genes involved in exopolysaccharide production and an activator for genes involved in stress response. To better understand the role of McbR in governing the switch from exponential growth to the biofilm state, we determined the crystal structure of McbR to 2.1 Å. The structure reveals McbR to be a member of the FadR C-terminal domain (FCD) family of the GntR superfamily of transcriptional regulators (this family was named after the first identified member, GntR, a transcriptional repressor of the gluconate operon of Bacillus subtilis). Previous to this study, only six of the predicted 2800 members of this family had been structurally characterized. Here, we identify the residues that constitute the McbR effector and DNA binding sites. In addition, comparison of McbR with other members of the FCD domain family shows that this family of proteins adopts highly distinct oligomerization interfaces, which has implications for DNA binding and regulation.

Direct Ras inhibitors identified from a structurally rigidified bicyclic peptide library

A one-bead-two-compound (OBTC) library of structurally rigidified bicyclic peptides was chemically synthesized on TentaGel microbeads (90 μm), with each bead displaying a unique bicyclic peptide on its surface and a linear encoding peptide of the same sequence in its interior. Screening of the library against oncogenic K-Ras G12V mutant identified two classes of Ras ligands. The class I ligands apparently bind to the effector-binding site and inhibit the Ras–Raf interaction, whereas the class II ligand appears to bind to a yet unidentified site different from the effector-binding site. These Ras ligands provide useful research tools and may be further developed into therapeutic agents.

Allosteric Regulation of the Human and Mouse Deoxyribonucleotide Triphosphohydrolase Sterile α-Motif/Histidine-Aspartate Domain-containing Protein 1 (SAMHD1)

The deoxyribonucleotide triphosphohydrolase SAMHD1 restricts lentiviral infection by depleting the dNTPs required for viral DNA synthesis. In cultured human fibroblasts SAMHD1 is expressed maximally during quiescence preventing accumulation of dNTPs outside S phase. siRNA silencing of SAMHD1 increases dNTP pools, stops cycling human cells in G1, and blocks DNA replication. Surprisingly, knock-out of the mouse gene does not affect the well being of the animals. dNTPs are both substrates and allosteric effectors for SAMHD1. In the crystal structure each subunit of the homotetrameric protein contains one substrate-binding site and two nonidentical effector-binding sites, site 1 binding dGTP, site 2 dGTP or dATP. Here we compare allosteric properties of pure recombinant human and mouse SAMHD1. Both enzymes are activated 3-4-fold by allosteric effectors. We propose that in quiescent cells where SAMHD1 is maximally expressed GTP binds to site 1 with very high affinity, stabilizing site 2 of the tetrameric structure. Any canonical dNTP can bind to site 2 and activate SAMHD1, but in cells only dATP or dTTP are present at sufficient concentrations. The apparent Km for dATP at site 2 is ∼10 μm for mouse and 1 μm for human SAMHD1, for dTTP the corresponding values are 50 and 2 μm. Tetrameric SAMHD1 is activated for the hydrolysis of any dNTP only after binding of a dNTP to site 2. The lower Km constants for human SAMHD1 induce activation at lower cellular concentrations of dNTPs thereby limiting the size of dNTP pools more efficiently in quiescent human cells.

A Transient Interaction between the Phosphate Binding Loop and Switch I Contributes to the Allosteric Network between Receptor and Nucleotide in Gαi1

Receptor-mediated activation of the Gα subunit of heterotrimeric G proteins requires allosteric communication between the receptor binding site and the guanine nucleotide binding site, which are separated by >30 Å. Structural changes in the allosteric network connecting these sites are predicted to be transient in the wild-type Gα subunit, making studies of these connections challenging. In the current work, site-directed mutants that alter the energy barriers between the activation states are used as tools to better understand the transient features of allosteric signaling in the Gα subunit. The observed differences in relative receptor affinity for intact Gαi1 subunits versus C-terminal Gαi1 peptides harboring the K345L mutation are consistent with this mutation modulating the allosteric network in the protein subunit. Measurement of nucleotide exchange rates, affinity for metarhodopsin II, and thermostability suggest that the K345L Gαi1 variant has reduced stability in both the GDP-bound and nucleotide-free states as compared with wild type but similar stability in the GTPγS-bound state. High resolution x-ray crystal structures reveal conformational changes accompanying the destabilization of the GDP-bound state. Of these, the conformation for Switch I was stabilized by an ionic interaction with the phosphate binding loop. Further site-directed mutagenesis suggests that this interaction between Switch I and the phosphate binding loop is important for receptor-mediated nucleotide exchange in the wild-type Gαi1 subunit.

Role of a PH Sensitive Site in Enac Na+ Self-Inhibition

The epithelial Na+ channel (ENaC) is key to the regulation of extracellular fluid volume and blood pressure through its action in the aldosterone-sensitive nephron. Inhibition of ENaC by extracellular Na+, referred to as Na+ self-inhibition, likely occurs through an allosteric mechanism of Na+ binding to low affinity sites in the large extracellular regions of ENaC subunits. The effector binding sites and transduction pathways remain unidentified. A recent comparative model of the ENaC α subunit revealed an acidic cleft, analogous to the acidic region observed in the resolved structure of acid sensing ion channel 1 (ASIC1). We hypothesized that this cleft and analogous clefts in the and subunits host Na+ effector sites. Mutations of acidic residues in the subunit acidic cleft led us to identify an aspartate on the loop connecting the central palm and β-ball domains that reaches towards the finger domain. We also identified nearby sites on the loop and other structures that affected Na+ self-inhibition. To determine whether these sites were involved in effector coordination, we measured the ability of mutants to change the effector specificity of Na+ self-inhibition, which is Na+ > Li+ >> K+ for wild type mouse ENaC. Mutation of the palm/-ball loop aspartate and several nearby sites weakened Na+ inhibition relative to Li+ and K+ inhibition. We hypothesized that protonating any carboxylate groups involved in Na+ coordination would weaken Na+ binding. Consistent with this, lowering the pH to 4.7 increased ENaC currents and reduced Na+ self-inhibition. Mutation of the palm/β-ball loop aspartate reversed pH activation of ENaC. Our results suggest that an aspartate in the acidic cleft of the subunit may coordinate Na+ for Na+ self-inhibition.

Simulations of the Structural Switch in PKM2 Mediating the Warburg Effect in Cancer

Pyruvate kinase activity is controlled by a tightly woven regulatory network. The oncofetal isoform of pyruvate kinase (PKM2) is a master regulator of cancer metabolism. PKM2 metabolically engages in parallel, feed‑forward, positive and negative feedback control as well as non‑metabolic functions as protein kinase and transcriptional coactivator contributing to cancer progression. Metabolically active PKM2 tetramer is allosterically regulated and responds to nutritional and stress signals, including 1,6-fructose-bis-phosphate. In contrast, the constitutively active PKM1 isoform of pyruvate kinase is not allosterically regulated, despite having more than 95% sequence identity to PKM2. To determine the molecular mechanism for allosteric regulation in PKM2 and reasons for its absence in PKM1, we performed replicate 100 ns-molecular dynamics simulations of tetrameric PKM1 and PKM2 isoforms with and without allosteric activator. Structural correlations within the PKM2 tetramer structure determine possible pathways mediating allosteric regulation as far as 1.5 nm between effector and enzymatic binding sites. A combined molecular dynamics and systems biology approach to PKM2 reveals how differences in biomolecular structure translate into a global rewiring of cancer metabolism.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Abstract A280: Small molecule inhibition of atypical protein kinase C iota reveals a functional basis for oncogenesis.

Abstract Background: Atypical protein kinase C iota (PKCι) is a serine/threonine kinase that has been implicated in a variety of cellular processes including polarity, migration, proliferation and survival. PKCι is amplified in many cancers correlating with poor prognosis, however the upstream activating inputs and downstream effectors are not well defined. Experimental Procedures: 3D MDCK epithelial cell spheroids were grown in Matrigel and used to assess polarized morphogenesis and growth. PKCι was manipulated by inhibition with CRT0066854, or transfection with siRNA or cDNA variants. A PKCι binding interface was identified by X-ray crystallography and interrogated by mass spectrometry of wildtype or binding mutant immunocomplexes. Results: To identify potential inputs into PKCι we assessed a panel of transformed MDCK cells; p110α, Src, Ras, ErbB2 and Raf. Parental MDCK cells form polarized cysts in Matrigel, however Ras, ErbB2 and p110α MDCK cells formed dysplastic spheroids. In the case of Ras and ErbB2, but not p110α MDCK spheroids, normal polarized morphogenesis could be rescued by siRNA-PKCι or partial PKCι inhibition with decreased spheroid size seen for all transformed cells lines. An effector binding site (PKCι RIPR motif) identified from the crystal structure of the kinase and characterized as a recruitment site for select targets including LLGL2 and Myosin X, was found to be critical for PKCι to confer polarized morphogenesis. Interestingly this binding motif shows a low frequency of mutation in a subset of human tumors. Conclusions: Downstream of Ras and ErbB2, PKCι activity is enhanced with a resulting dysplasia and loss of polarization, that can be rescued by PKCι inhibition. Mutation of the PKCι-RIPR effector binding motif that is found in cancer, prevents interaction with LLGL2 and its expression also elicits growth as non-polarized, dysplastic epithelial spheroids. This indicates that PKCι may promote cancer, not only by amplification, but by upstream oncogenic inputs and somatic mutation. The oncogene dependent PKCι hyperactivation is pro-proliferative in 3D culture, providing compelling evidence that PKCι is a good target for Ras mutant tumors. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A280. Citation Format: Mark Linch, Marta Sanz-Garcia, Svend Kjaer, Erika Soriano, Philippe Riou, Philip Knowles, Christian Dillon, Jon Roffey, Neil McDonald, Peter Parker. Small molecule inhibition of atypical protein kinase C iota reveals a functional basis for oncogenesis. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A280.

Engineering Plant Disease Resistance Based on TAL Effectors

Transcription activator-like (TAL) effectors are encoded by plant-pathogenic bacteria and induce expression of plant host genes. TAL effectors bind DNA on the basis of a unique code that specifies binding of amino acid residues in repeat units to particular DNA bases in a one-to-one correspondence. This code can be used to predict binding sites of natural TAL effectors and to design novel synthetic DNA-binding domains for targeted genome manipulation. Natural mechanisms of resistance in plants against TAL effector-containing pathogens have given insights into new strategies for disease control.

Lack of conventional oxygen-linked proton and anion binding sites does not impair allosteric regulation of oxygen binding in dwarf caiman hemoglobin.

In contrast to other vertebrate hemoglobins (Hbs) whose high intrinsic O2 affinities are reduced by red cell allosteric effectors (mainly protons, CO2, organic phosphates, and chloride ions), crocodilian Hbs exhibit low sensitivity to organic phosphates and high sensitivity to bicarbonate (HCO3(-)), which is believed to augment Hb-O2 unloading during diving and postprandial alkaline tides when blood HCO3(-) levels and metabolic rates increase. Examination of α- and β-globin amino acid sequences of dwarf caiman (Paleosuchus palpebrosus) revealed a unique combination of substitutions at key effector binding sites compared with other vertebrate and crocodilian Hbs: β82Lys→Gln, β143His→Val, and β146His→Tyr. These substitutions delete positive charges and, along with other distinctive changes in residue charge and polarity, may be expected to disrupt allosteric regulation of Hb-O2 affinity. Strikingly, however, P. palpebrosus Hb shows a strong Bohr effect, and marked deoxygenation-linked binding of organic phosphates (ATP and DPG) and CO2 as carbamate (contrasting with HCO3(-) binding in other crocodilians). Unlike other Hbs, it polymerizes to large complexes in the oxygenated state. The highly unusual properties of P. palpebrosus Hb align with a high content of His residues (potential sites for oxygenation-linked proton binding) and distinctive surface Cys residues that may form intermolecular disulfide bridges upon polymerization. On the basis of its singular properties, P. palpebrosus Hb provides a unique opportunity for studies on structure-function coupling and the evolution of compensatory mechanisms for maintaining tissue O2 delivery in Hbs that lack conventional effector-binding residues.

SPACER: server for predicting allosteric communication and effects of regulation

The SPACER server provides an interactive framework for exploring allosteric communication in proteins with different sizes, degrees of oligomerization and function. SPACER uses recently developed theoretical concepts based on the thermodynamic view of allostery. It proposes easily tractable and meaningful measures that allow users to analyze the effect of ligand binding on the intrinsic protein dynamics. The server shows potential allosteric sites and allows users to explore communication between the regulatory and functional sites. It is possible to explore, for instance, potential effector binding sites in a given structure as targets for allosteric drugs. As input, the server only requires a single structure. The server is freely available at http://allostery.bii.a-star.edu.sg/.

The transcriptional regulator CprK detects chlorination by combining direct and indirect readout mechanisms

The transcriptional regulator CprK controls the expression of the reductive dehalogenase CprA in organohalide-respiring bacteria. Desulfitobacterium hafniense CprA catalyses the reductive dechlorination of the terminal electron acceptor o-chlorophenol acetic acid, generating the phenol acetic acid product. It has been shown that CprK has ability to distinguish between the chlorinated CprA substrate and the de-halogenated end product, with an estimated an estimated 104-fold difference in affinity. Using a green fluorescent protein GFPUV-based transcriptional reporter system, we establish that CprK can sense o-chlorophenol acetic acid at the nanomolar level, whereas phenol acetic acid leads to transcriptional activation only when approaching micromolar levels. A structure–activity relationship study, using a range of o-chlorophenol acetic-acid-related compounds and key CprK mutants, combined with pKa calculations on the effector binding site, suggests that the sensitive detection of chlorination is achieved through a combination of direct and indirect readout mechanisms. Both the physical presence of the bulky chloride substituent as well as the accompanying electronic effects lowering the inherent phenol pKa are required for high affinity. Indeed, transcriptional activation by CprK appears strictly dependent on establishing a phenolate–K133 salt bridge interaction, rather than on the presence of a halogen atom per se. As K133 is strictly conserved within the CprK family, our data suggest that physiological function and future applications in biosensing are probably restricted to phenolic compounds.

Unraveling Hidden Regulatory Sites in Structurally Homologous Metalloproteases

Monitoring enzymatic activity in vivo of individual homologous enzymes such as the matrix metalloproteinases (MMPs) by antagonist molecules is highly desired for defining physiological and pathophysiological pathways. However, the rational design of antagonists targeting enzyme catalytic moieties specific to one of the homologous enzymes often appears to be an extremely difficult task. This is mainly due to the high structural homology at the enzyme active sites shared by members of the protein family. Accordingly, controlling enzymatic activity via alternative allosteric sites has become an attractive proposition for drug design targeting individual homologous enzymes. Yet, the challenge remains to identify such regulatory alternative sites that are often hidden and scattered over different locations on the protein's surface. We have designed branched amphiphilic molecules exhibiting specific inhibitory activity towards individual members of the MMP family. These amphiphilic isomers share the same chemical nature, providing versatile nonspecific binding reactivity that allows to probe hidden regulatory residues on a given protein surface. Using the advantage provided by amphiphilic ligands, here we explore a new approach for determining hidden regulatory sites. This approach includes diverse experimental analysis, such as structural spectroscopic analyses, NMR, and protein crystallography combined with computational prediction of effector binding sites. We demonstrate how our approach works by analyzing members of the MMP family that possess a unique set of such sites. Our work provides a proof of principle for using ligand effectors to unravel hidden regulatory sites specific to members of the structurally homologous MMP family. This approach may be exploited for the design of novel molecular effectors and therapeutic agents affecting protein catalytic function via interactions with structure-specific regulatory sites.

Specific Conformational States of Ras GTPase upon Effector Binding

To uncover the structural and dynamical determinants involved in the highly specific binding of Ras GTPase to its effectors, the conformational states of Ras in uncomplexed form and complexed to the downstream effectors Byr2, PI3Kγ, PLCε, and RalGDS were investigated using molecular dynamics and cross-comparison of the trajectories. The subtle changes in the dynamics and conformations of Ras upon effector binding require an analysis that targets local changes independent of global motions. Using a structural alphabet, a computational procedure is proposed to quantify local conformational changes. Positions detected by this approach were characterized as either specific for a particular effector, specific for an effector domain type, or as effector unspecific. A set of nine structurally connected residues (Ras residues 5–8, 32–35, 39–42, 55–59, 73–78, and 161–165), which link the effector binding site to the distant C-terminus, changed dynamics upon effector binding, indicating a potential effector-unspecific signaling route within the Ras structure. Additional conformational changes were detected along the N-terminus of the central β-sheet. Besides the Ras residues at the effector interface (e.g., D33, E37, D38, and Y40), which adopt effector-specific local conformations, the binding signal propagates from the interface to distant hot-spot residues, in particular to Y5 and D57. The results of this study reveal possible conformational mechanisms for the stabilization of the active state of Ras upon downstream effector binding and for the structural determinants responsible for effector specificity.

Addition of transcription activator‐like effector binding sites to a pathogen strain‐specific rice bacterial blight resistance gene makes it effective against additional strains and against bacterial leaf streak

Xanthomonas transcription activator-like (TAL) effectors promote disease in plants by binding to and activating host susceptibility genes. Plants counter with TAL effector-activated executor resistance genes, which cause host cell death and block disease progression. We asked whether the functional specificity of an executor gene could be broadened by adding different TAL effector binding elements (EBEs) to it. We added six EBEs to the rice Xa27 gene, which confers resistance to strains of the bacterial blight pathogen Xanthomonas oryzae pv. oryzae (Xoo) that deliver the TAL effector AvrXa27. The EBEs correspond to three other effectors from Xoo strain PXO99(A) and three from strain BLS256 of the bacterial leaf streak pathogen Xanthomonas oryzae pv. oryzicola (Xoc). Stable integration into rice produced healthy lines exhibiting gene activation by each TAL effector, and resistance to PXO99(A) , a PXO99(A) derivative lacking AvrXa27, and BLS256, as well as two other Xoo and 10 Xoc strains virulent toward wildtype Xa27 plants. Transcripts initiated primarily at a common site. Sequences in the EBEs were found to occur nonrandomly in rice promoters, suggesting an overlap with endogenous regulatory sequences. Thus, executor gene specificity can be broadened by adding EBEs, but caution is warranted because of the possible coincident introduction of endogenous regulatory elements.

Activation of p115-RhoGEF Requires Direct Association of Gα13 and the Dbl Homology Domain

RGS-containing RhoGEFs (RGS-RhoGEFs) represent a direct link between the G(12) class of heterotrimeric G proteins and the monomeric GTPases. In addition to the canonical Dbl homology (DH) and pleckstrin homology domains that carry out the guanine nucleotide exchange factor (GEF) activity toward RhoA, these RhoGEFs also possess RGS homology (RH) domains that interact with activated α subunits of G(12) and G(13). Although the GEF activity of p115-RhoGEF (p115), an RGS-RhoGEF, can be stimulated by Gα(13), the exact mechanism of the stimulation has remained unclear. Using combined studies with small angle x-ray scattering, biochemistry, and mutagenesis, we identify an additional binding site for activated Gα(13) in the DH domain of p115. Small angle x-ray scattering reveals that the helical domain of Gα(13) docks onto the DH domain, opposite to the surface of DH that binds RhoA. Mutation of a single tryptophan residue in the α3b helix of DH reduces binding to activated Gα(13) and ablates the stimulation of p115 by Gα(13). Complementary mutations at the predicted DH-binding site in the αB-αC loop of the helical domain of Gα(13) also affect stimulation of p115 by Gα(13). Although the GAP activity of p115 is not required for stimulation by Gα(13), two hydrophobic motifs in RH outside of the consensus RGS box are critical for this process. Therefore, the binding of Gα(13) to the RH domain facilitates direct association of Gα(13) to the DH domain to regulate its exchange activity. This study provides new insight into the mechanism of regulation of the RGS-RhoGEF and broadens our understanding of G protein signaling.

TAL Effector-Nucleotide Targeter (TALE-NT) 2.0: tools for TAL effector design and target prediction

Transcription activator-like (TAL) effectors are repeat-containing proteins used by plant pathogenic bacteria to manipulate host gene expression. Repeats are polymorphic and individually specify single nucleotides in the DNA target, with some degeneracy. A TAL effector-nucleotide binding code that links repeat type to specified nucleotide enables prediction of genomic binding sites for TAL effectors and customization of TAL effectors for use in DNA targeting, in particular as custom transcription factors for engineered gene regulation and as site-specific nucleases for genome editing. We have developed a suite of web-based tools called TAL Effector-Nucleotide Targeter 2.0 (TALE-NT 2.0; https://boglab.plp.iastate.edu/) that enables design of custom TAL effector repeat arrays for desired targets and prediction of TAL effector binding sites, ranked by likelihood, in a genome, promoterome or other sequence of interest. Search parameters can be set by the user to work with any TAL effector or TAL effector nuclease architecture. Applications range from designing highly specific DNA targeting tools and identifying potential off-target sites to predicting effector targets important in plant disease.

Study and reengineering of the binding sites and allosteric regulation of biosynthetic threonine deaminase by isoleucine and valine in Escherichia coli

Biosynthetic threonine deaminase (TD) is a key enzyme for the synthesis of isoleucine which is allosterically inhibited and activated by Ile and Val, respectively. The binding sites of Ile and Val and the mechanism of their regulations in TD are not clear, but essential for a rational design of efficient productive strain(s) for Ile and related amino acids. In this study, structure-based computational approach and site-directed mutagenesis were combined to identify the potential binding sites of Ile and Val in Escherichia coli TD. Our results demonstrated that each regulatory domain of the TD monomer possesses two nonequivalent effector-binding sites. The residues R362, E442, G445, A446, Y369, I460, and S461 only interact with Ile while E347, G350, and F352 are involved not only in the Ile binding but also in the Val binding. By further considering enzyme kinetic data, we propose a concentration-dependent mechanism of the allosteric regulation of TD by Ile and Val. For the construction of Ile overproducing strain, a novel TD mutant with double mutation of F352A/R362F was also created, which showed both higher activity and much stronger resistance to Ile inhibition comparing to those of wild-type enzyme. Overexpression of this mutant TD in E. coli JW3591 significantly increased the production of ketobutyrate and Ile in comparison to the reference strains overexpressing wild-type TD or the catabolic threonine deaminase (TdcB). This work builds a solid basis for the reengineering of TD and related microorganisms for Ile production.

Identification of New Ligands for the Methionine Biosynthesis Transcriptional Regulator (MetJ) by FAC-MS

We have developed a high-throughput approach using frontal affinity chromatography coupled to mass spectrometry (FAC-MS) for the identification and characterization of the small molecules that modulate transcriptional regulator (TR) binding to TR targets. We tested this approach using the methionine biosynthesis regulator (MetJ). We used effector mixtures containing S-adenosyl-L-methionine (SAM) and S-adenosyl derivatives as potential ligands for MetJ binding. The differences in the elution time of different compounds allowed us to rank the binding affinity of each compound. Consistent with previous results, FAC-MS showed that SAM binds to MetJ with the highest affinity. In addition, adenine and 5′-deoxy-5′-(methylthio)adenosine bind to the effector binding site on MetJ. Our experiments with MetJ demonstrate that FAC-MS is capable of screening complex mixtures of molecules and identifying high-affinity binders to TRs. In addition, FAC-MS experiments can be used to discriminate between specific and nonspecific binding of the effectors as well as to estimate the dissociation constant (K_d) for effector-TR binding_.

An Exclusive α/β Code Directs Allostery in TetR–Peptide Complexes

The allosteric mechanism of one of the best characterized bacterial transcription regulators, tetracycline repressor (TetR), has recently been questioned. Tetracycline binding induces cooperative folding of TetR, as suggested by recent unfolding studies, rather than switching between two defined conformational states, namely a DNA-binding-competent conformation and a non-DNA-binding conformation. Upon ligand binding, a host of near-native multiconformational structures collapse into a single, highly stabilized protein conformation that is no longer able to bind DNA. Here, structure–function studies performed with four synthetic peptides that bind to TetR and mimic the function of low-molecular-weight effectors, such as tetracyclines, provide new means to discriminate between different allosteric models. Whereas two inducing peptides bind in an extended β-like conformation, two anti-inducing peptides form an α-helix in the effector binding site of TetR. This exclusive bimodal interaction mode coincides with two distinct overall conformations of TetR, namely one that is identical with induced TetR and one that mirrors the DNA-bound state of TetR. Urea-induced unfolding studies show no increase in thermodynamic stability for any of the peptide complexes, although fluorescence measurements demonstrate peptide binding to TetR. This strongly suggests that, at least for these peptide effectors, a classical two-state allosteric model best describes TetR function.