33 research outputs found
A lipid-dependent link between activity and oligomerization state of the M. tuberculosis SMR protein TBsmr
AbstractTBsmr is a secondary active multidrug transporter from Mycobacterium tuberculosis that transports a plethora of compounds including antibiotics and fluorescent dyes. It belongs to the small multidrug resistance (SMR) superfamily and is structurally and functionally related to E. coli EmrE. Of particular importance is the link between protein function, oligomeric state and lipid composition. By freeze fracture EM, we found three different size distributions in three different lipid environments for TBsmr indicating different oligomeric states. The link of these states with protein activity has been probed by fluorescence spectroscopy revealing significant differences. The drug binding site has been probed further by 19F-MAS NMR through chemical labeling of native cysteine residues showing a water accessible environment in agreement with the alternating access model
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Phosphatidylinositol-4,5-Biphosphate-Dependent Rearrangement of TRPV4 Cytosolic Tails Enables Channel Activation by Physiological Stimuli
Most transient receptor potential (TRP) channels are regulated by phosphatidylinositol-4,5-biphosphate (PIP), although the structural rearrangements occurring on PIP binding are currently far from clear. Here we report that activation of the TRP vanilloid 4 (TRPV4) channel by hypotonic and heat stimuli requires PIP binding to and rearrangement of the cytosolic tails. Neutralization of the positive charges within the sequence KRWRK, which resembles a phosphoinositide-binding site, rendered the channel unresponsive to hypotonicity and heat but responsive to 4α-phorbol 12,13-didecanoate, an agonist that binds directly to transmembrane domains. Similar channel response was obtained by depletion of PIP from the plasma membrane with translocatable phosphatases in heterologous expression systems or by activation of phospholipase C in native ciliated epithelial cells. PIP facilitated TRPV4 activation by the osmotransducing cytosolic messenger 5âČ-6â-epoxyeicosatrienoic acid and allowed channel activation by heat in inside-out patches. Protease protection assays demonstrated a PIP-binding site within the N-tail. The proximity of TRPV4 tails, analyzed by fluorescence resonance energy transfer, increased by depleting PIP mutations in the phosphoinositide site or by coexpression with protein kinase C and casein kinase substrate in neurons 3 (PACSIN3), a regulatory molecule that binds TRPV4 N-tails and abrogates activation by cell swelling and heat. PACSIN3 lacking the Bin-Amphiphysin-Rvs (F-BAR) domain interacted with TRPV4 without affecting channel activation or tail rearrangement. Thus, mutations weakening the TRPV4âPIP interacting site and conditions that deplete PIP or restrict access of TRPV4 to PIPâin the case of PACSIN3âchange tail conformation and negatively affect channel activation by hypotonicity and heat.Molecular and Cellular Biolog
Trapping of a polyketide synthase module after CâC bond formation reveals transient acyl carrier domain interactions
Modular polyketide synthases (PKSs) are giant assembly lines that produce an impressive range of biologically active compounds. However, our understanding of the structural dynamics of these megasynthases, specifically the delivery of acyl carrier protein (ACP)âbound building blocks to the catalytic site of the ketosynthase (KS) domain, remains severely limited. Using a multipronged structural approach, we report details of the interâdomain interactions after CâC bond formation in a chainâbranching module of the rhizoxin PKS. Mechanismâbased crosslinking of an engineered module was achieved using a synthetic substrate surrogate that serves as a Michael acceptor. The crosslinked protein allowed us to identify an asymmetric state of the dimeric protein complex upon CâC bond formation by cryoâelectron microscopy (cryoâEM). The possible existence of two ACP binding sites, one of them a potential âparking positionâ for substrate loading, was also indicated by AlphaFold2 predictions. NMR spectroscopy showed that a transient complex is formed in solution, independent of the linker domains, and photochemical crosslinking/mass spectrometry of the standalone domains allowed us to pinpoint the interdomain interaction sites. The structural insights into a branching PKS module arrested after CâC bond formation allows a better understanding of domain dynamics and provides valuable information for the rational design of modular assembly lines
Methionine in a protein hydrophobic core drives tight interactions required for assembly of spider silk
Web spiders connect silk proteins, so-called spidroins, into fibers of extraordinary toughness. The spidroin N-terminal domain (NTD) plays a pivotal role in this process: it polymerizes spidroins through a complex mechanism of dimerization. Here we analyze sequences of spidroin NTDs and find an unusually high content of the amino acid methionine. We simultaneously mutate all methionines present in the hydrophobic core of a spidroin NTD from a nursery web spiderâs dragline silk to leucine. The mutated NTD is strongly stabilized and folds at the theoretical speed limit. The structure of the mutant is preserved, yet its ability to dimerize is substantially impaired. We find that side chains of core methionines serve to mobilize the fold, which can thereby access various conformations and adapt the association interface for tight binding. Methionine in a hydrophobic core equips a protein with the capacity to dynamically change shape and thus to optimize its function
Methionine in a protein hydrophobic core drives tight interactions required for assembly of spider silk
Web spiders connect silk proteins, so-called spidroins, into fibers of extraordinary toughness. The spidroin N-terminal domain (NTD) plays a pivotal role in this process: it polymerizes spidroins through a complex mechanism of dimerization. Here we analyze sequences of spidroin NTDs and find an unusually high content of the amino acid methionine. We simultaneously mutate all methionines present in the hydrophobic core of a spidroin NTD from a nursery web spiderâs dragline silk to leucine. The mutated NTD is strongly stabilized and folds at the theoretical speed limit. The structure of the mutant is preserved, yet its ability to dimerize is substantially impaired. We find that side chains of core methionines serve to mobilize the fold, which can thereby access various conformations and adapt the association interface for tight binding. Methionine in a hydrophobic core equips a protein with the capacity to dynamically change shape and thus to optimize its function
Development of Reduced Peptide Bond Pseudopeptide Michael Acceptors for the Treatment of Human African Trypanosomiasis
Human African Trypanosomiasis (HAT) is an endemic protozoan disease widespread in the sub-Saharan region that is caused by T. b. gambiense and T. b. rhodesiense. The development of molecules targeting rhodesain, the main cysteine protease of T. b. rhodesiense, has led to a panel of inhibitors endowed with micro/sub-micromolar activity towards the protozoa. However, whilst impressive binding affinity against rhodesain has been observed, the limited selectivity towards the target still remains a hard challenge for the development of antitrypanosomal agents. In this paper, we report the synthesis, biological evaluation, as well as docking studies of a series of reduced peptide bond pseudopeptide Michael acceptors (SPR10-SPR19) as potential anti-HAT agents. The new molecules show K-i values in the low-micro/sub-micromolar range against rhodesain, coupled with k(2nd) values between 1314 and 6950 M-1 min(-1). With a few exceptions, an appreciable selectivity over human cathepsin L was observed. In in vitro assays against T. b. brucei cultures, SPR16 and SPR18 exhibited single-digit micromolar activity against the protozoa, comparable to those reported for very potent rhodesain inhibitors, while no significant cytotoxicity up to 70 mu M towards mammalian cells was observed. The discrepancy between rhodesain inhibition and the antitrypanosomal effect could suggest additional mechanisms of action. The biological characterization of peptide inhibitor SPR34 highlights the essential role played by the reduced bond for the antitrypanosomal effect. Overall, this series of molecules could represent the starting point for further investigations of reduced peptide bond-containing analogs as potential anti-HAT agent