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Proton-coupled oligopeptide transporters (POTs) couple the inward transport of di- or tripeptides with an inwardly directed transport of protons. Evidence from several studies of different POTs has pointed toward involvement of a highly conserved sequence motif, E1XXE2RFXYY (from here on referred to as E1XXE2R), located on Helix I, in interactions with the proton. In this study, we investigated the intracellular substrate accumulation by motif variants with all possible combinations of glutamate residues changed to glutamine and arginine changed to a tyrosine, the latter being a natural variant found in the Escherichia coli POT YjdL. We found that YjdL motif variants with E1XXE2R, E1XXE2Y, E1XXQ2Y, or Q1XXE2Y were able to accumulate peptide, whereas those with E1XXQ2R, Q1XXE2R, or Q1XXQ2Y were unable to accumulate peptide, and Q1XXQ2R abolished uptake. These results suggest a mechanism that involves swapping of an intramotif salt bridge, i.e. R-E2 to R-E1, which is consistent with previous structural studies. Molecular dynamics simulations of the motif variants E1XXE2R and E1XXQ2R support this mechanism. The simulations showed that upon changing conformation arginine pushes Helix V, through interactions with the highly conserved FYING motif, further away from the central cavity in what could be a stabilization of an inward facing conformation. As E2 has been suggested to be the primary site for protonation, these novel findings show how protonation may drive conformational changes through interactions of two highly conserved motifs., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Saccharomyces cerevisiae Esc2p is a member of a conserved family of proteins that contain small ubiquitin-like modifier (SUMO)-like domains. It has been implicated in transcriptional silencing and shown to interact with the silencing protein Sir2p in a two-hybrid analysis. However, little is known about how Esc2p regulates the structure of silent chromatin. We demonstrate here that ESC2 differentially regulates silent chromatin at telomeric, rDNA, and HM loci. Specifically, ESC2 is required for efficient telomeric silencing and Sir2p association with telomeric silent chromatin and for silencing and maintenance of silent chromatin structure at rDNA. On the other hand, ESC2 negatively regulates silencing at HML and HMR and destabilizes HML silent chromatin without affecting Sir2p association with chromatin. We present evidence that Esc2p is associated with both transcriptionally silent and active loci in the genome, and the abundance of Esc2p is not correlated with the chromatin state at a particular locus. Using affinity pull-down analyses, we show that Esc2p and Sir2p interact in vivo, and recombinant Esc2p and Sir2p interact directly. Moreover, we dissect Esc2p and identify a putative SUMO-binding motif that is necessary and sufficient for interacting with Sir2p and SUMO and is required for the function of Esc2p in transcriptional silencing.

The serotonin transporter (SERT) regulates extracellular levels of the neurotransmitter serotonin (5-hydroxytryptamine) in the brain by facilitating uptake of released 5-hydroxytryptamine into neuronal cells. SERT is the target for widely used antidepressant drugs, including imipramine, fluoxetine, and (S)-citalopram, which are competitive inhibitors of the transport function. Knowledge of the molecular details of the antidepressant binding sites in SERT has been limited due to lack of structural data on SERT. Here, we present a characterization of the (S)-citalopram binding pocket in human SERT (hSERT) using mutational and computational approaches. Comparative modeling and ligand docking reveal that (S)-citalopram fits into the hSERT substrate binding pocket, where (S)-citalopram can adopt a number of different binding orientations. We find, however, that only one of these binding modes is functionally relevant from studying the effects of 64 point mutations around the putative substrate binding site. The mutational mapping also identify novel hSERT residues that are crucial for (S)-citalopram binding. The model defines the molecular determinants for (S)-citalopram binding to hSERT and demonstrates that the antidepressant binding site overlaps with the substrate binding site.

The serotonin transporter (SERT) regulates extracellular levels of serotonin (5-hydroxytryptamine, 5HT) in the brain by transporting 5HT into neurons and glial cells. The human SERT (hSERT) is the primary target for drugs used in the treatment of emotional disorders, including depression. hSERT belongs to the solute carrier 6 family that includes a bacterial leucine transporter (LeuT), for which a high resolution crystal structure has become available. LeuT has proved to be an excellent model for human transporters and has advanced the understanding of solute carrier 6 transporter structure-function relationships. However, the precise structural mechanism by which antidepressants inhibit hSERT and the location of their binding pockets are still elusive. We have identified a residue (Ser-438) located within the 5HT-binding pocket in hSERT to be a critical determinant for the potency of several antidepressants, including the selective serotonin reuptake inhibitor citalopram and the tricyclic antidepressants imipramine, clomipramine, and amitriptyline. A conservative mutation of Ser-438 to threonine (S438T) selectively increased the K(i) values for these antidepressants up to 175-fold. The effects of introducing a protein methyl group into the 5HT-binding pocket by S438T were absent or reduced for analogs of these antidepressants lacking a single methyl group. This suggests that these antidepressants interact directly with Ser-438 during binding to hSERT, implying an overlapping localization of substrate- and inhibitor-binding sites in hSERT suggesting that antidepressants function by a mechanism that involves direct occlusion of the 5HT-binding site.

Saccharomyces cerevisiae linker histone Hho1p is not essential for cell viability, and very little is known about its function in vivo. We show that deletion of HHO1 (hho1Delta) suppresses the defect in transcriptional silencing caused by a mutation in the globular domain of histone H4. hho1Delta also suppresses the reduction in HML silencing by the deletion of SIR1 that is involved in the establishment of silent chromatin at HML. We further show that hho1Delta suppresses changes in silent chromatin structure caused by the histone H4 mutation and sir1Delta. These results suggest that HHO1 plays a negative role in transcriptionally silent chromatin. We also provide evidence that Hho1p hinders the de novo establishment of silent chromatin but does not affect the stability of preexistent silent chromatin. Unlike canonical linker histones in higher eukaryotes that have a single conserved globular domain, Hho1p possesses two globular domains. We show that the carboxyl-terminal globular domain of Hho1p is dispensable for its function, suggesting that the mode of Hho1p action is similar to that of canonical linker histones.

Thiomandelic acid is a simple, broad spectrum, and reasonably potent inhibitor of metallo-beta-lactamases, enzymes that mediate resistance to beta-lactam antibiotics. We report studies by NMR and perturbed angular correlation (PAC) spectroscopy of the mode of binding of the R and S enantiomers of thiomandelic acid, focusing on their interaction with the two metal ions in cadmium-substituted Bacillus cereus metallo-beta-lactamase. The 113Cd resonances are specifically assigned to the metals in the two individual sites on the protein by using 113Cd-edited 1H NMR spectra. Each enantiomer of thiomandelate produces large downfield shifts of both 113Cd resonances and changes in the PAC spectra, which indicate that they bind such that the thiol of the inhibitor bridges between the two metals. For R-thiomandelate, this is unambiguously confirmed by the observation of scalar coupling between Halpha of the inhibitor and both cadmium ions. The NMR and PAC spectra reveal that the two chiral forms of the inhibitor differ in the details of their coordination geometry. The complex with R-thiomandelate, but not that with the S-enantiomer, shows evidence in the PAC spectra of a dynamic process in the nanosecond time regime, the possible nature of which is discussed. The thiomandelate complex of the mononuclear enzyme can be detected only at low metal to enzyme stoichiometry; the relative populations of mononuclear and binuclear enzyme as a function of cadmium concentration provide clear evidence for positive cooperativity in metal ion binding in the presence of the inhibitor, in contrast to the negative cooperativity observed in the free enzyme.

The momP1 promoter of the bacteriophage Mu mom operon is an example of a weak promoter. It contains a 19-base pair suboptimal spacer between the -35 (ACCACA) and -10 (TAGAAT) hexamers. Escherichia coli RNA polymerase is unable to bind to momP1 on its own. DNA distortion caused by the presence of a run of six T nucleotides overlapping the 5' end of the -10 element might prevent RNA polymerase from binding to momP1. To investigate the influence of the T(6) run on momP1 expression, defined substitution mutations were introduced by site-directed mutagenesis. In vitro probing experiments with copper phenanthroline ((OP)(2)Cu) and DNase I revealed distinct differences in cleavage patterns among the various mutants; in addition, compared with the wild type, the mutants showed an increase (variable) in momP1 promoter activity in vivo. Promoter strength analyses were in agreement with the ability of these mutants to form open complexes as well as to produce momP1-specific transcripts. No significant role is attributed to the overlapping and divergently organized promoter, momP2, in the expression of momP1 activity, as determined by promoter disruption analysis. These data support the view that an intrinsic DNA distortion in the spacer region of momP1 acts in cis as a negative element in mom operon transcription. This is a novel mechanism of regulation of toxic gene expression.