Phosphate is an indispensable metabolite in a wide variety of cells and is involved in nucleotide and lipid synthesis, signaling, and chemical energy storage. Proton-coupled phosphate transporters ...within the major facilitator family are crucial for phosphate uptake in plants and fungi. Similar proton-coupled phosphate transporters have been found in different protozoan parasites that cause human diseases, in breast cancer cells with elevated phosphate demand, in osteoclast-like cells during bone reabsorption, and in human intestinal Caco2BBE cells for phosphate homeostasis. However, the mechanism of proton-driven phosphate transport remains unclear. Here, we demonstrate in a eukaryotic, high-affinity phosphate transporter from
(PiPT) that deprotonation of aspartate 324 (D324) triggers phosphate release. Quantum mechanics/molecular mechanics molecular dynamics simulations combined with free energy sampling have been employed here to identify the proton transport pathways from D324 upon the transition from the occluded structure to the inward open structure and phosphate release. The computational insights so gained are then corroborated by studies of D45N and D45E amino acid substitutions via mutagenesis experiments. Our findings confirm the function of the structurally predicted cytosolic proton exit tunnel and suggest insights into the role of the titratable phosphate substrate.
The aquaporin family of channels was defined based on the inhibition of water transport by mercurial compounds. Despite the important role of mercurials, little is known about the structural changes ...involved upon mercury binding leading to channel inhibition. To elucidate the mechanism we designed a mutant, T183C, of aquaporin Z (AqpZ) patterned after the known mercury-sensitive site of aquaporin 1 (AQP1) and determined the X-ray crystal structures of the unbound and mercury blocked states. Superposition of the two structures shows no conformational rearrangement upon mercury binding. In the blocked structure, there are two mercury sites, one bound to Cys183 and occluding the pore, and a second, also bound to the same cysteine but found buried in an interstitial cavity. To test the mechanism of blockade we designed a different mutant, L170C, to produce a more effective mercury block at the pore site. In a dose-response inhibition study, this mutant was 20 times more sensitive to mercury than wild-type AqpZ and four times more sensitive than T183C. The X-ray structure of L170C shows four mercury atoms at, or near, the pore site defined in the T183C structure and no structural change upon mercury binding. Thus, we elucidate a steric inhibition mechanism for this important class of channels by mercury.
The first structure of an ammonia channel from the Amt/MEP/Rh protein superfamily, determined to 1.35 angstrom resolution, shows it to be a channel that spans the membrane 11 times. Two structurally ...similar halves span the membrane with opposite polarity. Structures with and without ammonia or methyl ammonia show a vestibule that recruits$NH_{4}^{+}/NH_{3}$, a binding site for NH4
+, and a 20 angstrom-long hydrophobic channel that lowers the$NH_{4}^+ pK_a$to below 6 and conducts NH3. Favorable interactions for NH3are seen within the channel and use conserved histidines. Reconstitution of AmtB into vesicles shows that AmtB conducts uncharged NH3.
The mycolic acid layer of the Mycobacterium tuberculosis cell wall is essential for viability and virulence, and the enzymes responsible for its synthesis are targets for antimycobacterial drug ...development. Polyketide synthase 13 (Pks13) is a module encoding several enzymatic and transport functions that carries out the condensation of two different long-chain fatty acids to produce mycolic acids. We determined structures by cryogenic-electron microscopy of dimeric multi-enzyme Pks13 purified from mycobacteria under normal growth conditions, captured with native substrates. Structures define the ketosynthase (KS), linker and acyl transferase (AT) domains at 1.8 Å resolution and two alternative locations of the N-terminal acyl carrier protein. These structures suggest intermediate states on the pathway for substrate delivery to the KS domain. Other domains, visible at lower resolution, are flexible relative to the KS-AT core. The chemical structures of three bound endogenous long-chain fatty acid substrates were determined by electrospray ionization mass spectrometry.
Signal sequences target proteins for secretion from cells or for integration into cell membranes. As nascent proteins emerge from the ribosome, signal sequences are recognized by the signal ...recognition particle (SRP), which subsequently associates with its receptor (SR). In this complex, the SRP and SR stimulate each other's GTPase activity, and GTP hydrolysis ensures unidirectional targeting of cargo through a translocation pore in the membrane. To define the mechanism of reciprocal activation, we determined the 1.9 A structure of the complex formed between these two GTPases. The two partners form a quasi-two-fold symmetrical heterodimer. Biochemical analysis supports the importance of the extensive interaction surface. Complex formation aligns the two GTP molecules in a symmetrical, composite active site, and the 3'OH groups are essential for association, reciprocal activation and catalysis. This unique circle of twinned interactions is severed twice on hydrolysis, leading to complex dissociation after cargo delivery.
Human tRNA3(Lys) is the primer for reverse transcription of HIV; the 3' end is complementary to the primer-binding site on HIV RNA. The complementarity ends at the 18th base, A58, which in tRNA3(Lys) ...is modified to remove Watson-Crick pairing. Motivated to test the role of the modification in terminating the primer-binding sequence and thus limiting run-on transcription, we asked how the modification of RNA could be accomplished. tRNA m(1)A58 methyltransferase (m(1)A58 MTase) methylates N1 of A58, which is buried in the TΨC-loop of tRNA, from cofactor S-adenosyl-L-methionine. This conserved tRNA modification is essential for stability of initiator tRNA in Saccharomyces cerevisiae. Reported here, three structures of human tRNA m(1)A58 MTase in complex with human tRNA3(Lys) and the product S-adenosyl-L-homocysteine show a dimer of heterodimers in which each heterodimer comprises a catalytic chain, Trm61, and a homologous but noncatalytic chain, Trm6, repurposed as a tRNA-binding subunit that acts in trans; tRNAs bind across the dimer interface such that Trm6 from the opposing heterodimer brings A58 into the active site of Trm61. T-loop and D-loop are splayed apart showing how A58, normally buried in tRNA, becomes accessible for modification. This result has broad impact on our understanding of the mechanisms of modifying internal sites in folded tRNA. The structures serve as templates for design of inhibitors that could be used to test tRNA m(1)A58 MTase's impact on retroviral priming and transcription.
Multidrug resistance protein 4 (MRP4) is a broadly expressed ATP-binding cassette transporter that is unique among the MRP subfamily for transporting prostanoids, a group of signaling molecules ...derived from unsaturated fatty acids. To better understand the basis of the substrate selectivity of MRP4, we used cryogenic-electron microscopy to determine six structures of nanodisc-reconstituted MRP4 at various stages throughout its transport cycle. Substrate-bound structures of MRP4 in complex with PGE
, PGE
and the sulfonated-sterol DHEA-S reveal a common binding site that accommodates a diverse set of organic anions and suggest an allosteric mechanism for substrate-induced enhancement of MRP4 ATPase activity. Our structure of a catalytically compromised MRP4 mutant bound to ATP-Mg
is outward-occluded, a conformation previously unobserved in the MRP subfamily and consistent with an alternating-access transport mechanism. Our study provides insights into the endogenous function of this versatile efflux transporter and establishes a basis for MRP4-targeted drug design.
To fire action-potential-like electrical signals, the vacuole membrane requires the two-pore channel TPC1, formerly called SV channel. The TPC1/SV channel functions as a depolarization-stimulated, ...non-selective cation channel that is inhibited by luminal Ca2+. In our search for species-dependent functional TPC1 channel variants with different luminal Ca2+ sensitivity, we found in total three acidic residues present in Ca2+ sensor sites 2 and 3 of the Ca2+-sensitive AtTPC1 channel from Arabidopsis thaliana that were neutral in its Vicia faba ortholog and also in those of many other Fabaceae. When expressed in the Arabidopsis AtTPC1-loss-of-function background, wild-type VfTPC1 was hypersensitive to vacuole depolarization and only weakly sensitive to blocking luminal Ca2+. When AtTPC1 was mutated for these VfTPC1-homologous polymorphic residues, two neutral substitutions in Ca2+ sensor site 3 alone were already sufficient for the Arabidopsis At-VfTPC1 channel mutant to gain VfTPC1-like voltage and luminal Ca2+ sensitivity that together rendered vacuoles hyperexcitable. Thus, natural TPC1 channel variants exist in plant families which may fine-tune vacuole excitability and adapt it to environmental settings of the particular ecological niche.
Translational accuracy and efficiency depend upon modification of uridines in the tRNA anticodon stem loop (ASL) by a highly conserved pseudouridine synthase TruA. TruA specifically modifies uridines ...at positions 38, 39, and/or 40 of tRNAs with highly divergent sequences and structures through a poorly characterized mechanism that differs from previously studied RNA-modifying enzymes. The molecular basis for the site and substrate “promiscuity” was studied by determining the crystal structures of
E. coli TruA in complex with two different leucyl tRNAs in conjunction with functional assays and computer simulation. The structures capture three stages of the TruA•tRNA reaction, revealing the mechanism by which TruA selects the target site. We propose that TruA utilizes the intrinsic flexibility of the ASL for site promiscuity and also to select against intrinsically stable tRNAs to avoid their overstabilization through pseudouridylation, thereby maintaining the balance between the flexibility and stability required for its biological function.
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