Identification of a gephyrin-binding motif in the GDP/GTP exchange factor collybistin.

The brain-specific GDP/GTP exchange factor collybistin interacts with the receptor-anchoring protein gephyrin and activates the Rho-like GTPase Cdc42, which is known to regulate actin cytoskeleton dynamics. Alternative splicing creates two collybistin variants, I and II. In coexpression experiments, collybistin II has been shown to induce the formation of submembraneous gephyrin aggregates which cluster with hetero-oligomeric glycine receptors (GlyRs). Here we identified residues critical for interaction with gephyrin in the linker region between the SH3 and the DH domains of collybistin. Respective collybistin deletion mutants failed to bind gephyrin upon coexpression in heterologous cells, in GST pull-down assays and in the yeast two-hybrid system. Site-directed mutagenesis revealed polar amino acid residues as essential determinants of gephyrin binding. Furthermore, in vitro gephyrin bound simultaneously to both collybistin and the GlyR beta-subunit binding motif. Our data are consistent with collybistin-gephyrin interactions occuring during inhibitory postsynaptic membrane formation.     

Molecular basis of the γ-aminobutyric acid A receptor α3 subunit interaction with the clustering protein gephyrin

The multifunctional scaffolding protein gephyrin is a key player in the formation of the postsynaptic scaffold at inhibitory synapses, clustering both inhibitory glycine receptors (GlyRs) and selected GABA(A) receptor (GABA(A)R) subtypes. We report a direct interaction between the GABA(A)R α3 subunit and gephyrin, mapping reciprocal binding sites using mutagenesis, overlay, and yeast two-hybrid assays. This analysis reveals that critical determinants of this interaction are located in the motif FNIVGTTYPI in the GABA(A)R α3 M3-M4 domain and the motif SMDKAFITVL at the N terminus of the gephyrin E domain. GABA(A)R α3 gephyrin binding-site mutants were unable to co-localize with endogenous gephyrin in transfected hippocampal neurons, despite being able to traffic to the cell membrane and form functional benzodiazepine-responsive GABA(A)Rs in recombinant systems. Interestingly, motifs responsible for interactions with GABA(A)R α2, GABA(A)R α3, and collybistin on gephyrin overlap. Curiously, two key residues (Asp-327 and Phe-330) in the GABA(A)R α2 and α3 binding sites on gephyrin also contribute to GlyR β subunit-E domain interactions. However, isothermal titration calorimetry reveals a 27-fold difference in the interaction strength between GABA(A)R α3 and GlyR β subunits with gephyrin with dissociation constants of 5.3 μm and 0.2 μm, respectively. Taken together, these observations suggest that clustering of GABA(A)R α2, α3, and GlyRs by gephyrin is mediated by distinct mechanisms at mixed glycinergic/GABAergic synapses.     

Mass spectrometric analysis of glycine receptor-associated gephyrin splice variants

Gephyrin is an ubiquitously expressed protein that, in the nervous system, is essential for synaptic anchoring of glycine receptors (GlyRs) and major GABAA receptor subtypes. The binding of gephyrin to the GlyR depends on an amphipathic motif within the large intracellular loop of the GlyRbeta subunit. The mouse gephyrin gene consists of 30 exons. Ten of these exons, encoding cassettes of 5-40 amino acids, are subject to alternative splicing (C1-C7, C4'-C6'). Since one of the cassettes, C5', has recently been reported to exclude GlyRs from GABAergic synapses, we investigated which cassettes are found in gephyrin associated with the GlyR. Gephyrin variants were purified from rat spinal cord, brain, and liver by binding to the glutathione S-transferase-tagged GlyRbeta loop or copurified with native GlyR from spinal cord by affinity chromatography and analyzed by mass spectrometry. In addition to C2 and C6', already known to be prominent, C4 was found to be abundant in gephyrin from all tissues examined. The nonneuronal cassette C3 was easily detected in liver but not in GlyR-associated gephyrin from spinal cord. C5 was present in brain and spinal cord polypeptides, whereas C5' was coisolated mainly from liver. Notably C5'-containing gephyrin bound to the GlyRbeta loop, inconsistent with its proposed selectivity for GABAA receptors. Our data show that GlyR-associated gephyrin, lacking C3, but enriched in C4 without C5, differs from other neuronal and nonneuronal gephyrin isoforms.     

The trafficking proteins Vacuolar Protein Sorting 35 and Neurobeachin interact with the glycine receptor β-subunit

Inhibitory glycine receptors (GlyRs) are densely packed in the postsynaptic membrane due to a high-affinity interaction of their β-subunits with the scaffolding protein gephyrin. Here, we used an affinity-based proteomic approach to identify the trafficking proteins Vacuolar Protein Sorting 35 (Vps35) and Neurobeachin (Nbea) as novel GlyR β-subunit (GlyRβ) interacting proteins in rat brain. Recombinant Vps35 and a central fragment of Nbea bound to the large intracellular loop of GlyRβ in glutathione-S-transferase pull-downs; in addition, Vps35 displayed binding to gephyrin. Immunocytochemical staining of spinal cord sections revealed Nbea immunoreactivity apposed to and colocalizing with marker proteins of inhibitory synapses. Our data are consistent with roles of Vps35 and Nbea in the retrieval and post-Golgi trafficking of synaptic GlyRs and possibly other neurotransmitter receptors.     

Post-phosphorylation prolyl isomerisation of gephyrin represents a mechanism to modulate glycine receptors function

The microtubule binding protein gephyrin plays a prominent role in establishing and maintaining a high concentration of inhibitory glycine receptors juxtaposed to presynaptic releasing sites. Here, we show that endogenous gephyrin undergoes proline-directed phosphorylation, which is followed by the recruitment of the peptidyl-prolyl isomerase Pin1. The interaction between gephyrin and Pin1 is strictly dependent on gephyrin phosphorylation and requires serine-proline consensus sites encompassing the gephyrin proline-rich domain. Upon binding, Pin1 triggers conformational changes in the gephyrin molecule, thus enhancing its ability to bind the beta subunit of GlyRs. Consistently, a downregulation of GlyR clusters was detected in hippocampal neurons derived from Pin1 knockout mice, which was paralleled by a reduction in the amplitude of glycine-evoked currents. Our results suggest that phosphorylation-dependent prolyl isomerisation of gephyrin represents a mechanism for regulating GlyRs function.     

Deciphering the structural framework of glycine receptor anchoring by gephyrin

Glycine is the major inhibitory neurotransmitter in the spinal cord and brain stem. Gephyrin is required to achieve a high concentration of glycine receptors (GlyRs) in the postsynaptic membrane, which is crucial for efficient glycinergic signal transduction. The interaction between gephyrin and the GlyR involves the E-domain of gephyrin and a cytoplasmic loop located between transmembrane segments three and four of the GlyR beta subunit. Here, we present crystal structures of the gephyrin E-domain with and without the GlyR beta-loop at 2.4 and 2.7 A resolutions, respectively. The GlyR beta-loop is bound in a symmetric 'key and lock' fashion to each E-domain monomer in a pocket adjacent to the dimer interface. Structure-guided mutagenesis followed by in vitro binding and in vivo colocalization assays demonstrate that a hydrophobic interaction formed by Phe 330 of gephyrin and Phe 398 and Ile 400 of the GlyR beta-loop is crucial for binding.     

Reciprocal stabilization of glycine receptors and gephyrin scaffold proteins at inhibitory synapses

Postsynaptic scaffold proteins immobilize neurotransmitter receptors in the synaptic membrane opposite to presynaptic vesicle release sites, thus ensuring efficient synaptic transmission. At inhibitory synapses in the spinal cord, the main scaffold protein gephyrin assembles in dense molecule clusters that provide binding sites for glycine receptors (GlyRs). Gephyrin and GlyRs can also interact outside of synapses, where they form receptor-scaffold complexes. Although several models for the formation of postsynaptic scaffold domains in the presence of receptor-scaffold interactions have been advanced, a clear picture of the coupled dynamics of receptors and scaffold proteins at synapses is lacking. To characterize the GlyR and gephyrin dynamics at inhibitory synapses, we performed fluorescence time-lapse imaging after photoconversion to directly visualize the exchange kinetics of recombinant Dendra2-gephyrin in cultured spinal cord neurons. Immuno-immobilization of endogenous GlyRs with specific antibodies abolished their lateral diffusion in the plasma membrane, as judged by the lack of fluorescence recovery after photobleaching. Moreover, the cross-linking of GlyRs significantly reduced the exchange of Dendra2-gephyrin compared with control conditions, suggesting that the kinetics of the synaptic gephyrin pool is strongly dependent on GlyR-gephyrin interactions. We did not observe any change in the total synaptic gephyrin levels after GlyR cross-linking, however, indicating that the number of gephyrin molecules at synapses is not primarily dependent on the exchange of GlyR-gephyrin complexes. We further show that our experimental data can be quantitatively accounted for by a model of receptor-scaffold dynamics that includes a tightly interacting receptor-scaffold domain, as well as more loosely bound receptor and scaffold populations that exchange with extrasynaptic pools. The model can make predictions for single-molecule data such as typical dwell times of synaptic proteins. Taken together, our data demonstrate the reciprocal stabilization of GlyRs and gephyrin at inhibitory synapses and provide a quantitative understanding of their dynamic organization.     

Neuronal cotransport of glycine receptor and the scaffold protein gephyrin

The dynamics of postsynaptic receptor scaffold formation and remodeling at inhibitory synapses remain largely unknown. Gephyrin, which is a multimeric scaffold protein, interacts with cytoskeletal elements and stabilizes glycine receptors (GlyRs) and individual subtypes of gamma-aminobutyric acid A receptors at inhibitory postsynaptic sites. We report intracellular mobility of gephyrin transports packets over time. Gephyrin units enter and exit active synapses within several minutes. In addition to previous reports of GlyR-gephyrin interactions at plasma membranes, we show cosedimentation and coimmunoprecipitation of both proteins from vesicular fractions. Moreover, GlyR and gephyrin are cotransported within neuronal dendrites and further coimmunoprecipitate and colocalize with the dynein motor complex. As a result, the blockade of dynein function or dynein-gephyrin interaction, as well as the depolymerization of microtubules, interferes with retrograde gephyrin recruitment. Our data suggest a GlyR-gephyrin-dynein transport complex and support the concept that gephyrin-motor interactions contribute to the dynamic and activity-dependent rearrangement of postsynaptic GlyRs, a process thought to underlie the regulation of synaptic strength.     

Regulation of glycine receptor diffusion properties and gephyrin interactions by protein kinase C

Glycine receptors (GlyRs) can dynamically exchange between synaptic and extrasynaptic locations through lateral diffusion within the plasma membrane. Their accumulation at inhibitory synapses depends on the interaction of the β-subunit of the GlyR with the synaptic scaffold protein gephyrin. An alteration of receptor-gephyrin binding could thus shift the equilibrium between synaptic and extrasynaptic GlyRs and modulate the strength of inhibitory neurotransmission. Using a combination of dynamic imaging and biochemical approaches, we have characterised the molecular mechanism that links the GlyR-gephyrin interaction with GlyR diffusion and synaptic localisation. We have identified a protein kinase C (PKC) phosphorylation site within the cytoplasmic domain of the β-subunit of the GlyR (residue S403) that causes a reduction of the binding affinity between the receptor and gephyrin. In consequence, the receptor's diffusion in the plasma membrane is accelerated and GlyRs accumulate less strongly at synapses. We propose that the regulation of GlyR dynamics by PKC thus contributes to the plasticity of inhibitory synapses and may be involved in maladaptive forms of synaptic plasticity.     

Gephyrin-mediated γ-aminobutyric acid type A and glycine receptor clustering relies on a common binding site

Gephyrin is the major protein determinant for the clustering of inhibitory neurotransmitter receptors. Earlier analyses revealed that gephyrin tightly binds to residues 398-410 of the glycine receptor β subunit (GlyR β) and, as demonstrated only recently, also interacts with GABA(A) receptors (GABA(A)Rs) containing the α1, α2, and α3 subunits. Here, we dissect the molecular basis underlying the interactions between gephyrin and GABA(A)Rs containing these α-subunits and compare them to the crystal structure of the gephyrin-GlyR β complex. Biophysical and biochemical assays revealed that, in contrast to its tight interaction with GlyR β, gephyrin only loosely interacts with GABA(A)R α2, whereas it has an intermediate affinity for the GABA(A)R α1 and α3 subunits. Despite the wide variation in affinities and the low overall sequence homology among the identified receptor subunits, competition assays confirmed the receptor-gephyrin interaction to be a mutually exclusive process. Selected gephyrin point mutants that critically weaken complex formation with GlyR β also abolished the GABA(A)R α1 and α3 interactions. Additionally, we identified a common binding motif with two conserved aromatic residues that are central for gephyrin binding. Consistent with the biochemical data, mutations of the corresponding residues within the cytoplasmic domain of α2 subunit-containing GABA(A)Rs attenuated clustering of these receptors at postsynaptic sites in hippocampal neurons. Taken together, our experiments provide key insights regarding similarities and differences in the complex formation between gephyrin and GABA(A)Rs compared with GlyRs and, hence, the accumulation of these receptors at postsynaptic sites.     

Evaluation of the protein interfaces that form an intermolecular four-helix bundle as studied by computer simulation

Rational design of protein surface is important for creating higher order protein structures, but it is still challenging. In this study, we designed in silico the several binding interfaces on protein surfaces that allow a de novo protein–protein interaction to be formed. We used a computer simulation technique to find appropriate amino acid arrangements for the binding interface. The protein–protein interaction can be made by forming an intermolecular four-helix bundle structure, which is often found in naturally occurring protein subunit interfaces. As a model protein, we used a helical protein, YciF. Molecular dynamics simulation showed that a new protein–protein interaction is formed depending on the number of hydrophobic and charged amino acid residues present in the binding surfaces. However, too many hydrophobic amino acid residues present in the interface negatively affected on the binding. Finally, we found an appropriate arrangement of hydrophobic and charged amino acid residues that induces a protein–protein interaction through an intermolecular four-helix bundle formation.     

Proteomic Analysis of Glycine Receptor β Subunit (GlyRβ)-interacting Proteins: EVIDENCE FOR SYNDAPIN I REGULATING SYNAPTIC GLYCINE RECEPTORS*

Glycine receptors (GlyRs) mediate inhibitory neurotransmission in spinal cord and brainstem. They are clustered at inhibitory postsynapses via a tight interaction of their β subunits (GlyRβ) with the scaffolding protein gephyrin. In an attempt to isolate additional proteins interacting with GlyRβ, we performed pulldown experiments with rat brain extracts using a glutathione S-transferase fusion protein encompassing amino acids 378–455 of the large intracellular loop of GlyRβ as bait. This identified syndapin I (SdpI) as a novel interaction partner of GlyRβ that coimmunoprecipitates with native GlyRs from brainstem extracts. Both SdpI and SdpII bound efficiently to the intracellular loop of GlyRβ in vitro and colocalized with GlyRβ upon coexpression in COS-7 cells. The SdpI-binding site was mapped to a proline-rich sequence of 22 amino acids within the intracellular loop of GlyRβ. Deletion and point mutation analysis disclosed that SdpI binding to GlyRβ is Src homology 3 domain-dependent. In cultured rat spinal cord neurons, SdpI immunoreactivity was found to partially colocalize with marker proteins of inhibitory and excitatory synapses. When SdpI was acutely knocked down in cultured spinal cord neurons by viral miRNA expression, postsynaptic GlyR clusters were significantly reduced in both size and number. Similar changes in GlyR cluster properties were found in spinal cultures from SdpI-deficient mice. Our results are consistent with a role of SdpI in the trafficking and/or cytoskeletal anchoring of synaptic GlyRs.     

Glycine Receptor Complex Analysis Using Immunoprecipitation‐Blue Native Gel Electrophoresis‐Mass Spectrometry

The pentameric glycine receptor (GlyR), comprising the α1 and β subunits, is a major inhibitory ionotropic receptor in brainstem and spinal cord. GlyRs interact with gephyrin (GPHN), a scaffold protein that anchors the GlyR in the plasma membrane and enables it to form clusters in glycinergic postsynapses. Using an interaction proteomics approach, evidence of the ArfGEFs IQ motif and Sec7 domain 3 (IQSEC3) and IQ motif and Sec7 domain 2 (IQSEC2) as two novel synaptic proteins interacting with GlyR complexes is provided. When the affinity‐isolated GlyR complexes are fractionated by blue native gel electrophoresis and characterized by mass spectrometry, GlyR α1β‐GPHN appears as the most abundant complex with a molecular weight of ≈1 MDa, and GlyR α1β‐GPHN‐IQSEC3 as a minor protein complex of ≈1.2 MDa. A third GlyR α1β‐GPHN‐IQSEC2 complex exists at the lowest amount with a mass similar to the IQSEC3 containing complex. Using yeast two‐hybrid it is demonstrated that IQSEC3 interacts with the GlyR complex by binding to the GPHN G domain at the N‐terminal of the IQSEC3 IQ‐like domain. The data provide direct evidence of the interaction of IQSEC3 with GlyR‐GPHN complexes, underscoring a potential role of these ArfGEFs in the function of glycinergic synapses.     

Comprehensive identification of Arabidopsis thaliana MYB transcription factors interacting with R/B-like BHLH proteins

In-depth analysis of protein-protein interaction specificities of the MYB protein family of Arabidopsis thaliana revealed a conserved amino acid signature ([DE]Lx(2)[RK]x(3)Lx(6)Lx(3)R) as the structural basis for interaction between MYB and R/B-like BHLH proteins. The motif has successfully been used to predict new MYB/BHLH interactions for A. thaliana proteins, it allows to discriminate between even closely related MYB proteins and it is conserved amongst higher plants. In A. thaliana, the motif is shared by fourteen R2R3 MYB proteins and six 1R MYB proteins. It is located on helices 1 and 2 of the R3 repeat and forms a characteristic surface-exposed pattern of hydrophobic and charged residues. Single-site mutation of any amino acid of the signature impairs the interaction. Two particular amino acids have been determined to account for most of the interaction stability. Functional specificity of MYB/BHLH complexes was investigated in vivo by a transient DFR promoter activation assay. Residues stabilizing the MYB/BHLH interaction were shown to be critical for promoter activation. By virtue of proved and predicted interaction specificities, this study provides a comprehensive survey of the MYB proteins that interact with R/B-like BHLH proteins potentially involved in the TTG1-dependent regulatory interaction network. The results are discussed with respect to multi-functionality, specificity and redundancy of MYB and BHLH protein function.     

Molecular Determinants of PAM2 Recognition by the MLLE Domain of Poly(A)-Binding Protein

MLLE (previously known as PABC) is a peptide-binding domain that is found in poly(A)-binding protein (PABP) and EDD (E3 isolated by differential display), a HECT E3 ubiquitin ligase also known as HYD (hyperplastic discs tumor suppressor) or UBR5. The MLLE domain from PABP recruits various regulatory proteins and translation factors to poly(A) mRNAs through binding of a conserved 12 amino acid peptide motif called PAM2 (for PABP-interacting motif 2). Here, we determined crystal structures of the MLLE domain from PABP alone and in complex with PAM2 peptides from PABP-interacting protein 2. The structures provide a detailed view of hydrophobic determinants of the MLLE binding coded by PAM2 positions 3, 5, 7, 10, and 12 and reveal novel intermolecular polar contacts. In particular, the side chain of the invariant MLLE residue K580 forms hydrogen bonds with the backbone of PAM2 residues 5 and 7. The structures also show that peptide residues outside of the conserved PAM2 motif contribute to binding. Altogether, the structures provide a significant advance in understanding the molecular basis for the binding of PABP by PAM2-containing proteins involved in translational control, mRNA deadenylation, and other cellular processes.     

Mutating the charged residues in the binding pocket of cellular retinoic acid-binding protein simultaneously reduces its binding affinity to retinoic acid and increases its thermostability.

Three-dimensional modeling of the complex between retinoic acid-binding protein (CRABP) and retinoic acid suggests that binding of the ligand is mediated by interaction between the carboxyl group of retinoic acid and two charged amino acids (Arg-111 and Arg-131) whose side chains project into the barrel of the protein. To assess the contribution of these amino acids to protein-ligand interaction, amino acid substitutions were made by oligonucleotide-directed, site-specific mutagenesis. The wild-type and mutant proteins were expressed in E. coli and subsequently purified. Like wild-type CRABP, the mutant proteins are composed mainly of beta-strands as determined by circular dichroism in the presence and absence of ligand, and thus presumably are folded into the same compact barrel structure as the wild-type protein. Mutants in which Arg-111 and Arg-131 are replaced by glutamine bind retinoic acid with significantly lower affinity than the wild-type protein, arguing that these two residues indeed interact with the ligand. The mutant proteins are more resistant to thermal denaturation than wild-type CRABP in the absence of retinoic acid, but they are not as thermostable as the CRABP-retinoic acid complex. These data suggest a model for CRABP-retinoic acid interaction in which the repulsive forces between the positively-charged arginine residues provide conformational flexibility to the native protein for retinoic acid to enter the binding pocket. Elimination of the positively-charged pair of amino acids produces a protein that is more thermostable than wild-type CRABP but less effective at ligand-binding.     

Protein precipitation and denaturation by dimethyl sulfoxide

Solvent conditions play a major role in a wide range of physical properties of proteins in solution. Organic solvents, including dimethyl sulfoxide (DMSO), have been used to precipitate, crystallize and denature proteins. We have studied here the interactions of DMSO with proteins by differential refractometry and amino acid solubility measurements. The proteins used, i.e., ribonuclease, lysozyme, beta-lactoglobulin and chymotrypsinogen, all showed negative preferential DMSO binding, or preferential hydration, at low DMSO concentrations, where they are in the native state. As the DMSO concentration was increased, the preferential interaction changed from preferential hydration to preferential DMSO binding, except for ribonuclease. The preferential DMSO binding correlated with structural changes and unfolding of these proteins observed at higher DMSO concentrations. Amino acid solubility measurements showed that the interactions between glycine and DMSO are highly unfavorable, while the interactions of DMSO with aromatic and hydrophobic side chains are favorable. The observed preferential hydration of the native protein may be explained from a combination of the excluded volume effects of DMSO and the unfavorable interaction of DMSO with a polar surface, as manifested by the unfavorable interactions of DMSO with the polar uncharged glycine molecule. Such an unfavorable interaction of DMSO with the native protein correlates with the enhanced self-association and precipitation of proteins by DMSO. Conversely, the observed conformational changes at higher DMSO concentration are due to increased binding of DMSO to hydrophobic and aromatic side chains, which had been newly exposed on protein unfolding.     

A hydrophobic pocket in the active site of glycolytic aldolase mediates interactions with Wiskott-Aldrich syndrome protein

Aldolase plays essential catalytic roles in glycolysis and gluconeogenesis. However, aldolase is a highly abundant protein that is remarkably promiscuous in its interactions with other cellular proteins. In particular, aldolase binds to highly acidic amino acid sequences, including the C terminus of the Wiskott-Aldrich syndrome protein, an actin nucleation-promoting factor. Here we report the crystal structure of tetrameric rabbit muscle aldolase in complex with a C-terminal peptide of Wiskott-Aldrich syndrome protein. Aldolase recognizes a short, four-residue DEWD motif (residues 498-501), which adopts a loose hairpin turn that folds around the central aromatic residue, enabling its tryptophan side chain to fit into a hydrophobic pocket in the active site of aldolase. The flanking acidic residues in this binding motif provide further interactions with conserved aldolase active site residues Arg-42 and Arg-303, aligning their side chains and forming the sides of the hydrophobic pocket. The binding of Wiskott-Aldrich syndrome protein to aldolase precludes intramolecular interactions of its C terminus with its active site and is competitive with substrate as well as with binding by actin and cortactin. Finally, based on this structure, a novel naphthol phosphate-based inhibitor of aldolase was identified, and its structure in complex with aldolase demonstrated mimicry of the Wiskott-Aldrich syndrome protein-aldolase interaction. The data support a model whereby aldolase exists in distinct forms that regulate glycolysis or actin dynamics.     

Structural properties of carnation mottle virus p7 movement protein and its RNA-binding domain

Plant viral movement proteins (MPs) participate actively in the intra- and intercellular movement of RNA plant viruses to such an extent that MP dysfunction impairs viral infection. However, the molecular mechanism(s) of their interaction with cognate nucleic acids are not well understood, partly due to the lack of structural information. In this work, a protein dissection approach was used to gain information on the structural and RNA-binding properties of this class of proteins, as exemplified by the 61-amino acid residue p7 MP from carnation mottle virus (CarMV). Circular dichroism spectroscopy showed that CarMV p7 is an alpha/beta RNA-binding soluble protein. Using synthetic peptides derived from the p7 sequence, we have identified three distinct putative domains within the protein. EMSA showed that the central region, from residue 17 to 35 (represented by peptide p7(17-35)), is responsible for the RNA binding properties of CarMV p7. This binding peptide populates a nascent alpha-helix in water solution that is further stabilized in the presence of either secondary structure inducers, such as trifluoroethanol and monomeric SDS, or RNA (which also changes its conformation upon binding to the peptide). Thus, the RNA recognition appears to occur via an "adaptive binding" mechanism. Interestingly, the amino acid sequence and structural properties of the RNA-binding domain of p7 seem to be conserved among carmoviruses and some other RNA-binding proteins and peptides. The low conserved N terminus of p7 (peptide p7(1-16)) is unstructured in solution. In contrast, the highly conserved C terminus motif (peptide p7(40-61)) adopts a beta-sheet conformation in aqueous solution. Alanine scanning mutagenesis of the RNA-binding motif showed how selected positive charged amino acids are more relevant than others in the RNA binding process and how hydrophobic amino acid side chains would participate in the stabilization of the protein-RNA complex.     

Functional roles of amino acid residues involved in forming the alpha-helix-turn-alpha-helix operator DNA binding motif of Tet repressor from Tn10.

The Tn10 derived Tet repressor contains an amino acid segment with high homology to the alpha-helix-turn-alpha-helix motif (HTH) of other DNA binding proteins. The five most conserved amino acids in HTH are probably involved in structural formation of the motif. Their functional role was probed by saturation mutagenesis yielding 95 single amino acid replacement mutants of Tet repressor. Their binding efficiencies to tet operator were quantitatively determined in vivo. All functional mutants contain amino acid substitutions consistent with their proposed role in a HTH. In particular, only the two smallest amino acids (serine, glycine) can substitute a conserved alanine in the proposed first alpha-helix without loss of activity. The last position of the first alpha-helix, the second position in the turn, and the fourth position in the second alpha-helix require mostly hydrophobic residues. The proposed C-terminus of the first alpha-helix is supported by a more active asparagine compared to glutamine replacement mutant of the wt leucine residue. The turn is located close to the protein surface as indicated by functional lysine and arginine replacements for valine. A glycine residue at the first position in the turn can be replaced by any amino acid yielding mutants with at least residual tet operator affinity. A structural model of the HTH of Tet repressor is presented.