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Abstract Sinorhizobium melilotisenses nutrients and compounds exuded from alfalfa host roots and coordinates an excitation, termination, and adaptation pathway during chemotaxis. We investigated the role of the novelS. melilotichemotaxis protein CheT. While CheT and theEscherichia coliphosphatase CheZ share little sequence homology, CheT is predicted to possess an α‐helix with a DXXXQ phosphatase motif. Phosphorylation assays demonstrated that CheT dephosphorylates the phosphate‐sink response regulator, CheY1~P by enhancing its decay two‐fold but does not affect the motor response regulator CheY2~P. Isothermal Titration Calorimetry (ITC) experiments revealed that CheT binds to a phosphomimic of CheY1~P with a KDof 2.9 μM, which is 25‐fold stronger than its binding to CheY1. Dissimilar chemotaxis phenotypes of the ΔcheTmutant andcheTDXXXQ phosphatase mutants led to the hypothesis that CheT exerts additional function(s). A screen for potential binding partners of CheT revealed that it forms a complex with the methyltransferase CheR. ITC experiments confirmed CheT/CheR binding with a KDof 19 μM, and a SEC‐MALS analysis determined a 1:1 and 2:1 CheT/CheR complex formation. Although they did not affect each other's enzymatic activity, CheT binding to CheY1~P and CheR may serve as a link between signal termination and sensory adaptation.
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Elucidating molecular interactions of L-nucleotides with HIV-1 reverse transcriptase and mechanism of M184V-caused drug resistance
Abstract Emtricitabine (FTC) and lamivudine (3TC), containing an oxathiolane ring with unnatural (−)-stereochemistry, are widely used nucleoside reverse transcriptase inhibitors (NRTIs) in anti-HIV therapy. Treatment with FTC or 3TC primarily selects for the HIV-1 RT M184V/I resistance mutations. Here we provide a comprehensive kinetic and structural basis for inhibiting HIV-1 RT by (−)-FTC-TP and (−)-3TC-TP and drug resistance by M184V. (−)-FTC-TP and (−)-3TC-TP have higher binding affinities (1/Kd) for wild-type RT but slower incorporation rates than dCTP. HIV-1 RT ternary crystal structures with (−)-FTC-TP and (−)-3TC-TP corroborate kinetic results demonstrating that their oxathiolane sulfur orients toward the DNA primer 3′-terminus and their triphosphate exists in two different binding conformations. M184V RT displays greater (>200-fold)Kdfor theL-nucleotides and moderately higher (>9-fold)Kdfor theD-isomers compared to dCTP. The M184V RT structure illustrates how the mutation repositions the oxathiolane of (−)-FTC-TP and shifts its triphosphate into a non-productive conformation.
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- Award ID(s):
- 1856617
- PAR ID:
- 10154174
- Publisher / Repository:
- Nature Publishing Group
- Date Published:
- Journal Name:
- Communications Biology
- Volume:
- 2
- Issue:
- 1
- ISSN:
- 2399-3642
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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