Chaperone Recycling in Late-Stage Flagellar Assembly.

The flagellum is a sophisticated nanomachine responsible for motility in Gram-negative bacteria. Flagellar assembly is a strictly choreographed process, in which the motor and export gate are formed first, followed by the extracellular propeller structure. Extracellular flagellar components are escorted to the export gate by dedicated molecular chaperones for secretion and self-assembly at the apex of the emerging structure. The detailed mechanisms of chaperone-substrate trafficking at the export gate remain poorly understood. Here, we structurally characterized the interaction of Salmonella enterica late-stage flagellar chaperones FliT and FlgN with the export controller protein FliJ. Previous studies showed that FliJ is absolutely required for flagellar assembly since its interaction with chaperone-client complexes controls substrate delivery to the export gate. Our biophysical and cell-based data show that FliT and FlgN bind FliJ cooperatively, with high affinity and on specific sites. Chaperone binding completely disrupts the FliJ coiled-coil structure and alters its interactions with the export gate. We propose that FliJ aids the release of substrates from the chaperone and forms the basis of chaperone recycling during late-stage flagellar assembly.

Progress toward automated methyl assignments for methyl-TROSY applications.

Methyl-TROSY spectroscopy has extended the reach of solution-state NMR to supra-molecular machineries over 100 kDa in size. Methyl groups are ideal probes for studying structure, dynamics, and protein-protein interactions in quasi-physiological conditions with atomic resolution. Successful implementation of the methodology requires accurate methyl chemical shift assignment, and the task still poses a significant challenge in the field. In this work, we outline the current state of technology for methyl labeling, data collection, data analysis, and nuclear Overhauser effect (NOE)-based automated methyl assignment approaches. We present MAGIC-Act and MAGIC-View, two Python extensions developed as part of the popular NMRFAM-Sparky package, and MAGIC-Net a standalone structure-based network analysis program. MAGIC-Act conducts statistically driven amino acid typing, Leu/Val pairing guided by 3D HMBC-HMQC, and NOESY cross-peak symmetry checking. MAGIC-Net provides model-based NOE statistics to aid in selection of a methyl labeling scheme. The programs provide a versatile, semi-automated framework for rapid methyl assignment.

2'-O-Methylation can increase the abundance and lifetime of alternative RNA conformational states.

2'-O-Methyl (Nm) is a highly abundant post-transcriptional RNA modification that plays important biological roles through mechanisms that are not entirely understood. There is evidence that Nm can alter the biological activities of RNAs by biasing the ribose sugar pucker equilibrium toward the C3'-endo conformation formed in canonical duplexes. However, little is known about how Nm might more broadly alter the dynamic ensembles of flexible RNAs containing bulges and internal loops. Here, using NMR and the HIV-1 transactivation response (TAR) element as a model system, we show that Nm preferentially stabilizes alternative secondary structures in which the Nm-modified nucleotides are paired, increasing both the abundance and lifetime of low-populated short-lived excited states by up to 10-fold. The extent of stabilization increased with number of Nm modifications and was also dependent on Mg2+. Through phi-value analysis, the Nm modification also provided rare insights into the structure of the transition state for conformational exchange. Our results suggest that Nm could alter the biological activities of Nm-modified RNAs by modulating their secondary structural ensembles as well as establish the utility of Nm as a tool for the discovery and characterization of RNA excited state conformations.

Structural and Functional Characterization of Sulfonium Carbon-Oxygen Hydrogen Bonding in the Deoxyamino Sugar Methyltransferase TylM1.

The N-methyltransferase TylM1 from Streptomyces fradiae catalyzes the final step in the biosynthesis of the deoxyamino sugar mycaminose, a substituent of the antibiotic tylosin. The high-resolution crystal structure of TylM1 bound to the methyl donor S-adenosylmethionine (AdoMet) illustrates a network of carbon-oxygen (CH···O) hydrogen bonds between the substrate's sulfonium cation and residues within the active site. These interactions include hydrogen bonds between the methyl and methylene groups of the AdoMet sulfonium cation and the hydroxyl groups of Tyr14 and Ser120 in the enzyme. To examine the functions of these interactions, we generated Tyr14 to phenylalanine (Y14F) and Ser120 to alanine (S120A) mutations to selectively ablate the CH···O hydrogen bonding to AdoMet. The TylM1 S120A mutant exhibited a modest decrease in its catalytic efficiency relative to that of the wild type (WT) enzyme, whereas the Y14F mutation resulted in an approximately 30-fold decrease in catalytic efficiency. In contrast, site-specific substitution of Tyr14 by the noncanonical amino acid p-aminophenylalanine partially restored activity comparable to that of the WT enzyme. Correlatively, quantum mechanical calculations of the activation barrier energies of WT TylM1 and the Tyr14 mutants suggest that substitutions that abrogate hydrogen bonding with the AdoMet methyl group impair methyl transfer. Together, these results offer insights into roles of CH···O hydrogen bonding in modulating the catalytic efficiency of TylM1.

Water-Mediated Carbon-Oxygen Hydrogen Bonding Facilitates S-Adenosylmethionine Recognition in the Reactivation Domain of Cobalamin-Dependent Methionine Synthase.

The C-terminal domain of cobalamin-dependent methionine synthase (MetH) has an essential role in catalyzing the reactivation of the enzyme following the oxidation of its cobalamin cofactor. This reactivation occurs through reductive methylation of the cobalamin using S-adenosylmethionine (AdoMet) as the methyl donor. Herein, we examine the molecular recognition of AdoMet by the MetH reactivation domain utilizing structural, biochemical, and computational approaches. Crystal structures of the Escherichia coli MetH reactivation domain in complex with AdoMet, the methyl transfer product S-adenosylhomocysteine (AdoHcy), and the AdoMet analogue inhibitor sinefungin illustrate that the ligands exhibit an analogous conformation within the solvent-exposed substrate binding cleft of the enzyme. AdoMet binding is stabilized by an intramolecular sulfur-oxygen chalcogen bond between the sulfonium and carboxylate groups of the substrate and by water-mediated carbon-oxygen hydrogen bonding between the sulfonium cation and the side chains of Glu1097 and Glu1128 that bracket the substrate binding cleft. AdoMet and sinefungin exhibited similar binding affinities for the MetH reactivation domain, whereas AdoHcy displayed an affinity for the enzyme that was an order of magnitude lower. Mutations of Glu1097 and Glu1128 diminished the AdoMet/AdoHcy binding selectivity ratio to approximately 2-fold, underscoring the role of these residues in enabling the enzyme to discriminate between the substrate and product. Together, these findings indicate that Glu1097 and Glu1128 in MetH promote high-affinity recognition of AdoMet and that sinefungin and potentially other AdoMet-based methyltransferase inhibitors can abrogate MetH reactivation, which would result in off-target effects associated with alterations in methionine homeostasis and one-carbon metabolism.

Atomic structures of excited state A-T Hoogsteen base pairs in duplex DNA by combining NMR relaxation dispersion, mutagenesis, and chemical shift calculations.

NMR relaxation dispersion studies indicate that in canonical duplex DNA, Watson-Crick base pairs (bps) exist in dynamic equilibrium with short-lived low abundance excited state Hoogsteen bps. N1-methylated adenine (m1A) and guanine (m1G) are naturally occurring forms of damage that stabilize Hoogsteen bps in duplex DNA. NMR dynamic ensembles of DNA duplexes with m1A-T Hoogsteen bps reveal significant changes in sugar pucker and backbone angles in and around the Hoogsteen bp, as well as kinking of the duplex towards the major groove. Whether these structural changes also occur upon forming excited state Hoogsteen bps in unmodified duplexes remains to be established because prior relaxation dispersion probes provided limited information regarding the sugar-backbone conformation. Here, we demonstrate measurements of C3' and C4' spin relaxation in the rotating frame (R1ρ) in uniformly 13C/15N labeled DNA as sensitive probes of the sugar-backbone conformation in DNA excited states. The chemical shifts, combined with structure-based predictions using an automated fragmentation quantum mechanics/molecular mechanics method, show that the dynamic ensemble of DNA duplexes containing m1A-T Hoogsteen bps accurately model the excited state Hoogsteen conformation in two different sequence contexts. Formation of excited state A-T Hoogsteen bps is accompanied by changes in sugar-backbone conformation that allow the flipped syn adenine to form hydrogen-bonds with its partner thymine and this in turn results in overall kinking of the DNA toward the major groove. Results support the assignment of Hoogsteen bps as the excited state observed in canonical duplex DNA, provide an atomic view of DNA dynamics linked to formation of Hoogsteen bps, and lay the groundwork for a potentially general strategy for solving structures of nucleic acid excited states.

Resolving sugar puckers in RNA excited states exposes slow modes of repuckering dynamics.

Recent studies have shown that RNAs exist in dynamic equilibrium with short-lived low-abundance 'excited states' that form by reshuffling base pairs in and around non-canonical motifs. These conformational states are proposed to be rich in non-canonical motifs and to play roles in the folding and regulatory functions of non-coding RNAs but their structure proves difficult to characterize given their transient nature. Here, we describe an approach for determining sugar pucker conformation in RNA excited states through nuclear magnetic resonance measurements of C1΄ and C4΄ rotating frame spin relaxation (R1ρ) in uniformly 13C/15N labeled RNA samples. Application to HIV-1 TAR exposed slow modes of sugar repuckering dynamics at the µs and ms timescale accompanying transitions between non-helical (C2΄-endo) to helical (C3΄-endo) conformations during formation of two distinct excited states. In contrast, we did not obtain any evidence for slow sugar repuckering dynamics for nucleotides in a variety of structural contexts that do not undergo non-helical to helical transitions. Our results outline a route for significantly improving the conformational characterization of RNA excited states and suggest that slow modes of repuckering dynamics gated by transient changes in secondary structure are quite common in RNA.

Robust IR-based detection of stable and fractionally populated G-C+ and A-T Hoogsteen base pairs in duplex DNA.

Noncanonical G-C+ and A-T Hoogsteen base pairs can form in duplex DNA and play roles in recognition, damage repair, and replication. Identifying Hoogsteen base pairs in DNA duplexes remains challenging due to difficulties in resolving syn versus antipurine bases with X-ray crystallography; and size limitations and line broadening can make them difficult to characterize by NMR spectroscopy. Here, we show how infrared (IR) spectroscopy can identify G-C+ and A-T Hoogsteen base pairs in duplex DNA across a range of different structural contexts. The utility of IR-based detection of Hoogsteen base pairs is demonstrated by characterizing the first example of adjacent A-T and G-C+ Hoogsteen base pairs in a DNA duplex where severe broadening complicates detection with NMR.

31P-dephased, 13C-detected REDOR for NMR crystallography at natural isotopic abundance.

Typically, the process of NMR-based structure determination relies on accurately measuring a large number of internuclear distances to serve as restraints for simulated annealing calculations. In solids, the rotational-echo double-resonance (REDOR) experiment is a widely used approach to determine heteronuclear dipolar couplings corresponding to distances usually in the range of 1.5-8Å. A challenge in the interpretation of REDOR data is the degeneracy of symmetric subunits in an oligomer or equivalent molecules in a crystal lattice, which produce REDOR trajectories that depend explicitly on two or more distances instead of one. This degeneracy cannot be overcome by either spin dilution (for molecules containing 31P, 19F and other highly abundant nuclei) or selective pulses (in the case where there is chemical shift degeneracy). For small, crystalline molecules, such as phosphoserine, we demonstrate that as many as five inter-molecular distances must be considered to model 31P-dephased REDOR data accurately. We report excellent agreement between simulation and experiment once lattice couplings, 31P chemical shift anisotropy, and radio-frequency field inhomogeneity are all taken into account. We also discuss the systematic inaccuracies that may result from approximations that consider only the initial slope of the REDOR trajectory and/or that utilize a two- or three-spin system. Furthermore, we demonstrate the applicability of 31P-dephased REDOR for validation or refinement of candidate crystal structures and show that this approach is especially informative for NMR crystallography of 31P-containing molecules.

Amphotericin forms an extramembranous and fungicidal sterol sponge.

For over 50 years, amphotericin has remained the powerful but highly toxic last line of defense in treating life-threatening fungal infections in humans with minimal development of microbial resistance. Understanding how this small molecule kills yeast is thus critical for guiding development of derivatives with an improved therapeutic index and other resistance-refractory antimicrobial agents. In the widely accepted ion channel model for its mechanism of cytocidal action, amphotericin forms aggregates inside lipid bilayers that permeabilize and kill cells. In contrast, we report that amphotericin exists primarily in the form of large, extramembranous aggregates that kill yeast by extracting ergosterol from lipid bilayers. These findings reveal that extraction of a polyfunctional lipid underlies the resistance-refractory antimicrobial action of amphotericin and suggests a roadmap for separating its cytocidal and membrane-permeabilizing activities. This new mechanistic understanding is also guiding development of what are to our knowledge the first derivatives of amphotericin that kill yeast but not human cells.

Molecular determinants of phospholipid synergy in blood clotting.

Many regulatory processes in biology involve reversible association of proteins with membranes. Clotting proteins bind to phosphatidylserine (PS) on cell surfaces, but a clear picture of this interaction has yet to emerge. We present a novel explanation for membrane binding by GLA domains of clotting proteins, supported by biochemical studies, solid-state NMR analyses, and molecular dynamics simulations. The model invokes a single "phospho-L-serine-specific" interaction and multiple "phosphate-specific" interactions. In the latter, the phosphates in phospholipids interact with tightly bound Ca(2+) in GLA domains. We show that phospholipids with any headgroup other than choline strongly synergize with PS to enhance factor X activation. We propose that phosphatidylcholine and sphingomyelin (the major external phospholipids of healthy cells) are anticoagulant primarily because their bulky choline headgroups sterically hinder access to their phosphates. Following cell damage or activation, exposed PS and phosphatidylethanolamine collaborate to bind GLA domains by providing phospho-L-serine-specific and phosphate-specific interactions, respectively.

Atomic view of calcium-induced clustering of phosphatidylserine in mixed lipid bilayers.

Membranes play key regulatory roles in biological processes, with bilayer composition exerting marked effects on binding affinities and catalytic activities of a number of membrane-associated proteins. In particular, proteins involved in diverse processes such as vesicle fusion, intracellular signaling cascades, and blood coagulation interact specifically with anionic lipids such as phosphatidylserine (PS) in the presence of Ca(2+) ions. While Ca(2+) is suspected to induce PS clustering in mixed phospholipid bilayers, the detailed structural effects of this ion on anionic lipids are not established. In this study, combining magic angle spinning (MAS) solid-state NMR (SSNMR) measurements of isotopically labeled serine headgroups in mixed lipid bilayers with molecular dynamics (MD) simulations of PS lipid bilayers in the presence of different counterions, we provide site-resolved insights into the effects of Ca(2+) on the structure and dynamics of lipid bilayers. Ca(2+)-induced conformational changes of PS in mixed bilayers are observed in both liposomes and Nanodiscs, a nanoscale membrane mimetic of bilayer patches. Site-resolved multidimensional correlation SSNMR spectra of bilayers containing (13)C,(15)N-labeled PS demonstrate that Ca(2+) ions promote two major PS headgroup conformations, which are well resolved in two-dimensional (13)C-(13)C, (15)N-(13)C, and (31)P-(13)C spectra. The results of MD simulations performed on PS lipid bilayers in the presence or absence of Ca(2+) provide an atomic view of the conformational effects underlying the observed spectra.

Backbone 1H, 13C and 15N resonance assignments of the extracellular domain of tissue factor.

Backbone (1)H, (13)C and (15)N resonance assignments are presented for the extracellular domain of tissue factor. Tissue factor is the integral membrane protein that initiates blood coagulation through the formation an enzymatic complex with the plasma serine protease, factor VIIa.

Protein-phospholipid interactions in blood clotting.

Most steps of the blood clotting cascade require the assembly of a serine protease with its specific regulatory protein on a suitable phospholipid bilayer. Unfortunately, the molecular details of how blood clotting proteins bind to membrane surfaces remain poorly understood, owing to a dearth of techniques for studying protein-membrane interactions at high resolution. Our laboratories are tackling this question using a combination of approaches, including nanoscale membrane bilayers, solid-state NMR, and large-scale molecular dynamics simulations. These studies are now providing structural insights at atomic resolution into clotting protein-membrane interactions.