Determinants in the sequence specific binding of two plant transcription factors, CBF1 and NtERF2, to the DRE and GCC motifs

Arabidopsis ERF proteins such as DREB1, DREB2, and CBF1 bind to the dehydration-responsive element (DRE), which has the sequence TACCGACAT. Mutation analyses reveal that a central 5 bp CCGAC core of the DRE is the minimal sequence motif (designated as the DRE motif in this paper), to which the ERF domain fragment of CBF1 (CBF1-F) binds specifically with a binding K(d) at the nanomolar level. In contrast, the ERF domain fragment of the tobacco ERF2 (NtERF2-F) does not interact with the DRE motif, but restrictedly recognizes the sequence containing a minimal 6 bp GCCGCC motif (designated as the GCC motif in this paper). However, CBF1-F binds to the GCC motif with a binding activity similar to its binding activity for the DRE motif. These in vitro binding variations were further demonstrated through reporter cotransformation assays, suggesting that the DRE and GCC motifs are two similar sequence motifs sharing a common core region of CCGNC with a discriminating guanine base at the 5'-end of the GCC motif. Binding analyses with the mutated ERF domain show that such a unique binding of NtERF2-F to the GCC motif can be altered by the substitution of A14 with valine in beta-strand 2 of its ERF domain, the mutant NtERF2-F, ERFav, acquiring a binding to the DRE motif with a K(d) comparable to that for CBF1-F binding to the DRE motif. This demonstrates that A14 is an important determinant of the NtERF2-F binding specificity. A possible mechanism of the binding specificity determination is discussed.     

Structural elements of the osteopontin SVVYGLR motif important for the interaction with alpha(4) integrins

The osteopontin SVVYGLR motif binds the integrins alpha(4)beta(1) and alpha(9)beta(1). We show that alpha(4)beta(7) also interacts with this motif and that an SVVYGLR-OH peptide antagonises the alpha(4)beta(7) MAdCAM interaction. The important elements of this motif required to bind alpha(4)beta(1) and alpha(4)beta(7) were probed using a series of mutated peptides based around SVVYGLR. Leu167 is important for the interaction with alpha(4) integrins, as is the C-terminal carboxylic acid of Arg168 exposed by thrombin cleavage. The importance of the acidic group means that SVVYGLR has structural elements in common with other alpha(4) integrin-binding motifs and suggests why thrombin cleavage activates this motif.     

Two processivity clamp interactions differentially alter the dual activities of UmuC

DNA polymerases of the Y family promote survival by their ability to synthesize past lesions in the DNA template. One Escherichia coli member of this family, DNA pol V (UmuC), which is primarily responsible for UV-induced and chemically induced mutagenesis, possesses a canonical beta processivity clamp-binding motif. A detailed analysis of this motif in DNA pol V (UmuC) showed that mutation of only two residues in UmuC is sufficient to result in a loss of UV-induced mutagenesis. Increased levels of wild-type beta can partially rescue this loss of mutagenesis. Alterations in this motif of UmuC also cause loss of the cold-sensitive and beta-dependent synthetic lethal phenotypes associated with increased levels of UmuD and UmuC that are thought to represent an exaggeration of a DNA damage checkpoint. By designing compensatory mutations in the cleft between domains II and III in beta, we restored UV-induced mutagenesis by a UmuC beta-binding motif variant. A recent co-crystal structure of the 'little finger' domain of E. coli pol IV (DinB) with beta suggests that, in addition to the canonical beta-binding motif, a second site of pol IV ((303)VWP(305)) interacts with beta at the outer rim of the dimer interface. Mutational analysis of the corresponding motif in UmuC showed that it is dispensable for induced mutagenesis, but that alterations in this motif result in loss of the cold-sensitive phenotype. These two beta interaction sites of UmuC affect the dual functions of UmuC differentially and indicate subtle and sophisticated polymerase management by the beta clamp.     

Identity and functions of CxxC-derived motifs

Two cysteines separated by two other residues (the CxxC motif) are employed by many redox proteins for formation, isomerization, and reduction of disulfide bonds and for other redox functions. The place of the C-terminal cysteine in this motif may be occupied by serine (the CxxS motif), modifying the functional repertoire of redox proteins. Here we found that the CxxC motif may also give rise to a motif, in which the C-terminal cysteine is replaced with threonine (the CxxT motif). Moreover, in contrast to a view that the N-terminal cysteine in the CxxC motif always serves as a nucleophilic attacking group, this residue could also be replaced with threonine (the TxxC motif), serine (the SxxC motif), or other residues. In each of these CxxC-derived motifs, the presence of a downstream alpha-helix was strongly favored. A search for conserved CxxC-derived motif/helix patterns in four complete genomes representing bacteria, archaea, and eukaryotes identified known redox proteins and suggested possible redox functions for several additional proteins. Catalytic sites in peroxiredoxins were major representatives of the TxxC motif, whereas those in glutathione peroxidases represented the CxxT motif. Structural assessments indicated that threonines in these enzymes could stabilize catalytic thiolates, suggesting revisions to previously proposed catalytic triads. Each of the CxxC-derived motifs was also observed in natural selenium-containing proteins, in which selenocysteine was present in place of a catalytic cysteine.     

An ion-responsive motif in the second transmembrane segment of rhodopsin-like receptors

A L(M)xxxD(N, E) motif (x=a non-ionic amino acid residue, most frequently A, S, L or F; small capitals indicating a minor representation) is found in the second transmembrane (tm2) segment of most G-protein coupling metazoan receptors of the rhodopsin family (Rh-GPCRs). Changes in signal transduction, agonist binding and receptor cycling are known for numerous receptors bearing evolved or experimentally introduced mutations in this tm2 motif, especially of its aspartate residue. The [Na⁺] sensitivity of the receptor-agonist interaction relates to this aspartate in a number of Rh-GPCRs. Native non-conservative mutations in the tm2 motif only rarely coincide with significant changes in two other ubiquitous features of the rhodopsin family, the seventh transmembrane N(D)PxxY(F) motif and the D(E)RY(W,F) or analogous sequence at the border of the third transmembrane helix and the second intracellular loop. Native tm2 mutations with Rh-GPCRs frequently result in constitutive signaling, and with visual opsins also in shifts to short-wavelength sensitivity. Substitution of a strongly basic residue for the tm2 aspartate in Taste-2 receptors could be connected to a lack of sodium sensing by these receptors. These properties could be consistent with ionic interactions, and even of ion transfer, that involve the tm2 motif. A decrease in cation sensing by this motif is usually connected to an enhanced constitutive interaction of the mutated receptors with cognate G- proteins, and also relates to both the constitutive and the overall activity of the short-wavelength opsins.     

The identification of a minimal dimerization motif QXXS that enables homo- and hetero-association of transmembrane helices in vivo

Assembly of transmembrane (TM) domains is a critical step in the function of membrane proteins, and therefore, determining the amino acid motifs that mediate this process is important. Studies along this line have shown that the GXXXG motif is involved in TM assembly. In this study we characterized the minimal dimerization motif in the bacterial Tar-1 homodimer TM domain, which does not contain a GXXXG sequence. We found that a short polar motif QXXS is sufficient to induce stable TM-TM interactions. Statistical analysis revealed that this motif appears to be significantly over-represented in a bacterial TM data base compared with its theoretical expectancy, suggesting a general role for this motif in TM assembly. A truncated short TM peptide (9 residues) that contains the QXXS motif interacted slightly with the wild-type Tar-1. However, the same short TM peptide regained wild-type-like activity when conjugated to an octanoyl aliphatic moiety. Biophysical studies indicated that this modification compensated for the missing hydrophobicity, stabilized alpha-helical structure, and enabled insertion of the peptide into the membrane core. These findings serve as direct evidence that even a short peptide containing a minimal recognition motif is sufficient to inhibit the proper assembly of TM domains. Interestingly, electron microscopy revealed that above the critical micellar concentration, the TM lipopeptide forms a network of nanofibers, which can serve for the slow release of the active lipopeptide.     

Mutational analysis of the leucine zipper-like motif of the human immunodeficiency virus type 1 envelope transmembrane glycoprotein.

The N-terminal region of the envelope (env) transmembrane protein of human immunodeficiency virus type 1 (HIV-1) has a leucine zipper-like motif. This highly conserved zipper motif, which consists of a heptad repeat of leucine or isoleucine residues, has been suggested to play a role in HIV-1 env glycoprotein oligomerization. This hypothesis was tested by replacing the highly conserved leucine or isoleucine residues in the zipper motif with a strong alpha-helix breaker, proline. We report here that such substitutions did not abolish the ability of env protein to form oligomers, indicating that this highly conserved zipper motif does not have a crucial role in env protein oligomerization. However, the mutant viruses all showed impaired infectivity, suggesting that this conserved zipper motif can have an important role in the virus life cycle.     

Sequential determination of ligands binding to discrete components in heterogeneous mixtures by iterative panning and blocking (IPAB)

Biopanning has been used extensively in conjunction with purified components, but there are also examples in which mixtures of targets have been investigated. This study introduces a methodological innovation, termed iterative panning and blocking (IPAB), to extend the range of specific interactions that can be probed in mixtures. Here this procedure is used to probe a mixture of high molecular mass components of human cord blood with phage-peptide display libraries. The initial panning recovered phage that bore the consensus motif Gly-Pro-Arg-Pro, a known fibrinogen-binding motif. These phage bound specifically to purified fibrinogen. A series of peptides containing the Gly-Pro-Arg-Pro motif efficiently blocked the binding of phage having the same motif, presumably by binding to their common target. A second round of panning was performed against the same target mixture in the presence of this blocking peptide. Phage recovered from this second panning exhibited a motif (Ser-His-Tyr) that was subsequently shown to bind specifically to complement component C1q. A second peptide containing this motif specifically blocked the interaction of the phage with C1q. A third round of panning performed in the presence of both the fibrinogen- and the C1q- blocking peptides yielded phage with a new peptide motif (Asn-Pro-Phe) that also bound specifically to C1q, apparently at a new site. The three motifs isolated through this iterative process were distinct in that each was blocked only by its corresponding peptide. This IPAB strategy can be applied to many high diversity selection procedures that target complex mixtures.     

Identification of a novel di-leucine motif mediating K(+)/Cl(-) cotransporter KCC2 constitutive endocytosis

The neuron-specific potassium-chloride cotransporter 2 (KCC2) plays a crucial role, by controlling chloride extrusion, in the development and maintenance of inhibitory neurotransmission. Although it is now well established that activity-dependent mechanisms can down regulate KCC2 gene expression, the role of post-translational mechanisms in controlling KCC2 expression, specifically at the cell-surface, are poorly understood. We therefore set out to identify the mechanisms and motifs regulating KCC2 endocytosis, one important pathway that may control KCC2 membrane expression. Using a fluorescence-based assay, we show KCC2 when expressed in HEK293 cells is constitutively internalized via a dynamin- and clathrin-dependent pathway. Consistent with this, we demonstrate KCC2 from adult mouse brain associates in vivo with the clathrin-binding adaptor protein-2 (AP-2) complex. Using an endocytosis reporter system, we identify the presence of an autonomous endocytosis motif in the carboxyl cytoplasmic terminus of KCC2. By site-directed mutagenesis we define this novel KCC2 endocytic motif as a non-canonical di-leucine motif, (657)LLXXEE(662). Finally by mutating this motif in the context of full-length KCC2 we demonstrate that this novel KCC2 endocytic motif is essential for the constitutive internalization of KCC2 and for binding to the AP-2 complex. Subsequent sequence analysis reveals this motif is highly conserved between the closely related K(+)/Cl(-) family members that mediate chloride efflux, but absent from the more distant related cotransporters controlling chloride influx. In conclusion, our results indicate constitutive internalization of KCC2 is clathrin-mediated and dependent on the binding of AP-2 to this novel endocytic motif. Furthermore, that this process appears to be an evolutionarily conserved mechanism amongst functionally homologous cotransporters.     

Octamer motif is required for the NF-kappaB-mediated induction of the inducible nitric oxide synthase gene expression in RAW 264.7 macrophages

The promoter of the mouse inducible nitric oxide synthase (iNOS) has a putative octamer motif (ATGCAAAA) which exists 24 bp upstream from the TATA box and is mismatched at a single residue from the consensus octamer motif. To examine whether this site is involved in iNOS expression, we constructed various deletions and site-directed mutants of the iNOS promoter linked to the chloramphenicol acetyltransferase (CAT) reporter gene, transfected the constructs into RAW 264.7 macrophages, and stimulated the cells with interferon-gamma (IFN-gamma) and/or lipopolysaccharide (LPS). CAT activity was not induced by LPS in constructs containing only the octamer motif (-71 to +82), but was induced with constructs containing the octamer motif and the upstream sequences of the NF-kappaB site (-91 to +82). However, a site-directed mutation of the octamer motif in the context of the -91 to +82 promoter construct or an extended promoter construct (-1542 to +82) abolished IFN-gamma and/or LPS-induced CAT activity. Similar results were obtained from site-directed mutants at either the NF-kappaB site or both the NF-kappaB site and octamer motif in these two constructs. In addition, we demonstrated that the conversion of the iNOS octamer motif into a consensus sequence increased CAT activity. Electrophoretic mobility shift assay (EMSA) performed with the NF-kappaB site or the octamer motif-containing oligonucleotide probe revealed that NF-kappaB binding was induced by LPS treatment, while the Oct-1 binding was constitutive. Competition assays performed with octamer-related oligonucleotide competitors derived from the immunoglobulin-kappaB or SV40 promoter confirmed the identity of the iNOS promoter sequence as being a Oct-1 binding site. EMSA carried out using a probe containing both the NF-kappaB site and the octamer motif identified two LPS-induced complexes. Competition assays with each NF-kappaB site or octamer motif competitor revealed that NF-kappaB and Oct-1 were present in these two complexes. These data suggest that, besides the NF-kappaB site, the octamer motif is essential for the maximal expression of the iNOS gene in murine macrophages, and the direct interaction of Oct-1 and NF-kappaB is important for the regulation of this gene.     

A short motif in the C-terminus of mouse bestrophin 3 [corrected] inhibits its activation as a Cl channel

Bestrophins are a new family of anion channels. Here, we examined the Cl channel activity of mBest4. Surprisingly, wild type mouse bestrophin-4 (mBest4) did not induce functional Cl channels when over-expressed in HEK293 cells. However, deletion of part of the C-terminus (residues 353-669) produced large Cl currents, suggesting the presence of a C-terminal motif that inhibited Cl channel function. Deletion of a short motif (356-364) or substitution of certain residues in this motif with alanines also resulted in expression of robust Cl currents. The channel activity of the mBest4 protein lacking the C-terminus (residues 353-669) was specifically inhibited by co-expression of C-terminal fragments of mBest4 having the inhibitory motif, suggesting that the C-terminal motif blocked mBest4 channel activity probably by interacting with the channel pore.     

A motif present in the main cytoplasmic loop of nicotinic acetylcholine receptors and catalases

A motif containing five conserved amino acids (RXPXTH(X)14P) was detected in 111 proteins, including 82 nicotinic acetylcholine receptor (nAChR) subunits and 20 catalases. To explore possible functional roles of this motif in nAChRs two approaches were used: first, the motif sequences in nAChR subunits and catalases were analysed and compared; and, second, deletions in the rat alpha2 and beta4 nAChR subunits expressed in Xenopus oocytes were analysed. Compared to the three-dimensional structure of bovine hepatic catalase, structural coincidences were found in the motif of catalases and nAChRs. On the other hand, partial deletions of the motif in the alpha2 or beta4 subunits and injection of the mutants into oocytes was followed by a very weak expression of functional nAChRs; oocytes injected with alpha2 and beta4 subunits in which the entire motif had been deleted failed to elicit any acetylcholine currents. The results suggest that the motif may play a role in the activation of nAChRs.     

The cytoplasmic tail of the severe acute respiratory syndrome coronavirus spike protein contains a novel endoplasmic reticulum retrieval signal that binds COPI and promotes interaction with membrane protein

Like other coronaviruses, severe acute respiratory syndrome coronavirus (SARS CoV) assembles at and buds into the lumen of the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC). Accumulation of the viral envelope proteins at this compartment is a prerequisite for virus assembly. Previously, we reported the identification of a dibasic motif (KxHxx) in the cytoplasmic tail of the SARS CoV spike (S) protein that was similar to a canonical dilysine ER retrieval signal. Here we demonstrate that this motif is a novel and functional ER retrieval signal which reduced the rate of traffic of the full-length S protein through the Golgi complex. The KxHxx motif also partially retained two different reporter proteins in the ERGIC region and reduced their rates of trafficking, although the motif was less potent than the canonical dilysine signal. The dibasic motif bound the coatomer complex I (COPI) in an in vitro binding assay, suggesting that ER retrieval may contribute to the accumulation of SARS CoV S protein near the virus assembly site for interaction with other viral structural proteins. In support of this, we found that the dibasic motif on the SARS S protein was required for its localization to the ERGIC/Golgi region when coexpressed with SARS membrane (M) protein. Thus, the cycling of SARS S through the ER-Golgi system may be required for its incorporation into assembling virions in the ERGIC.     

Mechanisms of endoplasmic-reticulum export of glycine transporter-1 (GLYT1)

The GLYT1 (glycine transporter-1) regulates both glycinergic and glutamatergic neurotransmission by controlling the reuptake of glycine at synapses. Trafficking to the cell surface of GLYT1 is critical for its function. In the present paper, by using mutational analysis of the GLYT1 C-terminal domain, we identified the evolutionarily conserved motif R(575)L(576)(X(8))D(585) as being necessary for ER (endoplasmic reticulum) export. This is probably due to its capacity to bind Sec24D, a component of the COPII (coatomer coat protein II) complex. This ER export motif was active when introduced into the related GLYT2 transporter but not in the unrelated VSVG (vesicular-stomatitis virus glycoprotein)-GLYT1 protein in which this motif was mutated but was not transported to the plasma membrane, although this effect was rescued by co-expressing these mutants with wild-type GLYT1. This behaviour suggests that GLYT1 might form oligomers along the trafficking pathway. Cross-linking assays performed in rat brain synaptosomes and FRET (fluorescence resonance energy transfer) microscopy in living cells confirmed the existence of GLYT1 oligomers. In summary, we have identified a motif involved in the ER exit of GLYT1 and, in analysing the influence of this motif, we have found evidence that oligomerization is important for the trafficking of GLYT1 to the cell surface. Because this motif is conserved in the NSS (sodium- and chloride-dependent neurotransmitter transporter) family, it is possible that this finding could be extrapolated to other related transporters.     

Structural insight into modest binding of a non-PXXP ligand to the signal transducing adaptor molecule-2 Src homology 3 domain

Although some exceptional motifs have been identified, it is well known that the PXXP motif is the motif of ligand proteins generally recognized by the Src homology 3 (SH3) domain. SH3-ligand interactions are usually weak, with ordinary KD approximately 10 microM. The structural basis for a tight and specific association (KD = 0.24 microm) between Gads SH3 and a novel motif, PX(V/I)(D/N)RXXKP, was revealed in a previous structural analysis of the complex formed between them. In this paper, we report the crystal structure of the signal transducing adaptor molecule-2 (STAM2) SH3 domain in complex with a peptide with a novel motif derived from a ligand protein, UBPY. The derived KD value for this complex is 27 microM. The notable difference in affinity for these parallel complexes may be explained because the STAM2 SH3 structure does not provide a specificity pocket for binding, whereas the Gads SH3 structure does. Instead, the structure of STAM2 SH3 is analogous to that of Grb2 SH3 which, in addition to normal PXXP ligands, has also been shown to moderately recognize the novel motif discussed herein. Thus, the extremely tight interaction observed between Gads SH3 and the novel motif is caused not by an innate ability of the novel motif but rather by an evolutionary change in the Gads SH3 domain. Instead, SH3 domains of STAM2 and Grb2 retain the moderate characteristics of recognizing their ligand proteins like other SH3 domains for appropriate transient interactions between signaling molecules.