NMR chemical shift assignment of Drosophila odorant binding protein 44a in complex with 8(Z)-eicosenoic acid.

The odorant binding protein, OBP44a is one of the most abundant proteins expressed in the brain of the developing fruit fly Drosophila melanogaster. Its cellular function has not yet been determined. The OBP family of proteins is well established to recognize hydrophobic molecules. In this study, NMR is employed to structurally characterize OBP44a. NMR chemical shift perturbation measurements confirm that OBP44a binds to fatty acids. Complete assignments of the backbone chemical shifts and secondary chemical shift analysis demonstrate that the apo state of OBP44a is comprised of six α-helices. Upon binding 8(Z)-eicosenoic acid (8(Z)-C20:1), the OBP44a C-terminal region undergoes a conformational change, from unstructured to α-helical. In addition to C-terminal restructuring upon ligand binding, some hydrophobic residues show dramatic chemical shift changes. Surprisingly, several charged residues are also strongly affected by lipid binding. Some of these residues could represent key structural features that OBP44a relies on to perform its cellular function. The NMR chemical shift assignment is the first step towards characterizing the structure of OBP44a and how specific residues might play a role in lipid binding and release. This information will be important in deciphering the biological function of OBP44a during fly brain development.

Co-crystal structures of the fluorogenic aptamer Beetroot show that close homology may not predict similar RNA architecture.

Beetroot is a homodimeric in vitro selected RNA that binds and activates DFAME, a conditional fluorophore derived from GFP. It is 70% sequence-identical to the previously characterized homodimeric aptamer Corn, which binds one molecule of its cognate fluorophore DFHO at its interprotomer interface. We have now determined the Beetroot-DFAME co-crystal structure at 1.95 Å resolution, discovering that this RNA homodimer binds two molecules of the fluorophore, at sites separated by ~30 Å. In addition to this overall architectural difference, the local structures of the non-canonical, complex quadruplex cores of Beetroot and Corn are distinctly different, underscoring how subtle RNA sequence differences can give rise to unexpected structural divergence. Through structure-guided engineering, we generated a variant that has a 12-fold fluorescence activation selectivity switch toward DFHO. Beetroot and this variant form heterodimers and constitute the starting point for engineered tags whose through-space inter-fluorophore interaction could be used to monitor RNA dimerization.

Many dissimilar NusG protein domains switch between α-helix and ß-sheet folds.

Folded proteins are assumed to be built upon fixed scaffolds of secondary structure, α-helices and ß-sheets. Experimentally determined structures of >58,000 non-redundant proteins support this assumption, though it has recently been challenged by ~100 fold-switching proteins. Though ostensibly rare, these proteins raise the question of how many uncharacterized proteins have shapeshifting-rather than fixed-secondary structures. Here, we use a comparative sequence-based approach to predict fold switching in the universally conserved NusG transcription factor family, one member of which has a 50-residue regulatory subunit experimentally shown to switch between α-helical and ß-sheet folds. Our approach predicts that 24% of sequences in this family undergo similar α-helix ⇌ ß-sheet transitions. While these predictions cannot be reproduced by other state-of-the-art computational methods, they are confirmed by circular dichroism and nuclear magnetic resonance spectroscopy for 10 out of 10 sequence-diverse variants. This work suggests that fold switching may be a pervasive mechanism of transcriptional regulation in all kingdoms of life.

Tsg101/ESCRT-I recruitment regulated by the dual binding modes of K63-linked diubiquitin.

The ESCRT-I protein Tsg101 plays a critical role in viral budding and endocytic sorting. Although Tsg101 is known to recognize monoubiquitin (Ub1), here we show that it can also bind several diubiquitins (K48-Ub2, N-Ub2, and K63-Ub2), with a preference for K63-linked Ub2. The NMR structure of the Tsg101:K63-Ub2 complex showed that while the Ub1-binding site accommodates the distal domain of Ub2, the proximal domain alternatively binds two different sites, the vestigial active site and an N-terminal helix. Mutation of each site results in distinct phenotypes regarding the recruitment of Tsg101 partners. Mutation in the vestigial active site abrogates interaction between Tsg101 and the HIV-1 protein Gag but not Hrs, a cellular protein. Mutation at the N-terminal helix alters Gag but not Hrs-Tsg101 localization. Given the broad involvement of Tsg101 in diverse cellular functions, this discovery advances our understanding of how the ESCRT protein recognizes binding partners and sorts endocytic cargo.

Simultaneous measurement of 1HC/N-R2's for rapid acquisition of backbone and sidechain paramagnetic relaxation enhancements (PREs) in proteins.

Paramagnetic relaxation enhancements (PREs) are routinely used to provide long-range distance restraints for the determination of protein structures, to resolve protein dynamics, ligand-protein binding sites, and lowly populated species, using Nuclear Magnetic Resonance Spectroscopy (NMR). Here, we propose a simultaneous 1H-15 N, 1H-13C SESAME based pulse scheme for the rapid acquisition of 1HC/N-R2 relaxation rates for the determination of backbone and sidechain PREs of proteins. The 1HN-R2 rates from the traditional and our approach on Ubiquitin (UBQ) are well correlated (R2 = 0.99), revealing their potential to be used quantitatively. Comparison of the S57C UBQ calculated and experimental PREs provided backbone and side chain Q factors of 0.23 and 0.24, respectively, well-fitted to the UBQ NMR structure, showing that our approach can be used to acquire accurate PRE rates from the functionally important sites of proteins but in at least half the time as traditional methods.

Squeezing lipids: NMR characterization of lipoprotein particles under pressure.

Determining the particle size and number of lipoprotein components found in blood plasma (HDL, LDL and VLDL) has become an important clinical tool in diagnosing risk of cardiovascular disease. Proton (1H) NMR spectroscopy methods to quantify lipoprotein particle subclasses have been advancing since NMR lineshape analysis of plasma samples was first proposed in the 1990's. NMR methods, including a more recent DOSY-based diffusion spectroscopy test, provide the foundation for the advanced lipoprotein tests, including Lipoprotein® and Liposcale® analyses available for clinical use to determine particle size and number. At the time of this submission, no NMR studies exist which explore physical parameters of individual lipoprotein fractions when they are deformed by pressure. This study reports 1H NMR frequency shifts and T2* measurements for the broad methyl peak attributed to terminal methyls (cholesteryl positions 26, 27 and terminal acyl methyl groups) in three primary lipoprotein fractions as a function of hydraulic pressure. This terminal CH3 resonance shifted linearly upfield as a function of pressure for HDL and VLDL (observed slopes of -0.014 Hz/bar). The LDL terminal CH3 resonance shows segmented behavior, with a shallow slope between 0-900 bar (-0.008 hz/bar) and a slope similar to HDL and VDL across the range from 1000 to 2400 bar (slope -0.016 Hz/bar). 1H T2* values measured for VLDL and HDL dropped linearly with increasing pressure. 1H T2* values for LDL demonstrated segmented behavior as a function of pressure. The unique behavior observed for LDL terminal CH3 frequency and 1H T2* trends suggests an approximate pressure at which phase transition occurs.

The Kindlin Outside Connection.

Transmembrane integrin bridges the extracellular and intracellular environments and is activated by focal adhesion proteins, talin and kindlin. Activated integrin engages ligands from the extracellular matrix and controls intracellular responses. In this issue of Structure, Zhu et al. (2019) describe an initial step involving recruitment of paxillin by ubiquitin-like kindlin-2 domain.

The Structure of Melanoregulin Reveals a Role for Cholesterol Recognition in the Protein's Ability to Promote Dynein Function.

Melanoregulin (Mreg) is a small, highly charged, multiply palmitoylated protein present on the membrane of melanosomes. Mreg is implicated in the transfer of melanosomes from melanocytes to keratinocytes, and in promoting the microtubule minus end-directed transport of these organelles. The possible molecular function of Mreg was identified by solving its structure using nuclear magnetic resonance (NMR) spectroscopy. Mreg contains six α helices forming a fishhook-like fold in which positive and negative charges occupy opposite sides of the protein's surface and sandwich a putative, cholesterol recognition sequence (CRAC motif). Mreg containing a point mutation within its CRAC motif still targets to late endosomes/lysosomes, but no longer promotes their microtubule minus end-directed transport. Moreover, wild-type Mreg does not promote the microtubule minus end-directed transport of late endosomes/lysosomes in cells transiently depleted of cholesterol. Finally, reversing the charge of three clustered acidic residues partially inhibits Mreg's ability to drive these organelles to microtubule minus ends.

Solution structure of the cap-independent translational enhancer and ribosome-binding element in the 3' UTR of turnip crinkle virus.

The 3(') untranslated region (3(') UTR) of turnip crinkle virus (TCV) genomic RNA contains a cap-independent translation element (CITE), which includes a ribosome-binding structural element (RBSE) that participates in recruitment of the large ribosomal subunit. In addition, a large symmetric loop in the RBSE plays a key role in coordinating the incompatible processes of viral translation and replication, which require enzyme progression in opposite directions on the viral template. To understand the structural basis for the large ribosomal subunit recruitment and the intricate interplay among different parts of the molecule, we determined the global structure of the 102-nt RBSE RNA using solution NMR and small-angle x-ray scattering. This RNA has many structural features that resemble those of a tRNA in solution. The hairpins H1 and H2, linked by a 7-nucleotide linker, form the upper part of RBSE and hairpin H3 is relatively independent from the rest of the structure and is accessible to interactions. This global structure provides insights into the three-dimensional layout for ribosome binding, which may serve as a structural basis for its involvement in recruitment of the large ribosomal subunit and the switch between viral translation and replication. The experimentally determined three-dimensional structure of a functional element in the 3(') UTR of an RNA from any organism has not been previously reported. The RBSE structure represents a prototype structure of a new class of RNA structural elements involved in viral translation/replication processes.

A method for helical RNA global structure determination in solution using small-angle x-ray scattering and NMR measurements.

We report a "top-down" method that uses mainly duplexes' global orientations and overall molecular dimension and shape restraints, which were extracted from experimental NMR and small-angle X-ray scattering data, respectively, to determine global architectures of RNA molecules consisting of mostly A-form-like duplexes. The method is implemented in the G2G (from global measurement to global structure) toolkit of programs. We demonstrate the efficiency and accuracy of the method by determining the global structure of a 71-nt RNA using experimental data. The backbone root-mean-square deviation of the ensemble of the calculated global structures relative to the X-ray crystal structure is 3.0+/-0.3 A using the experimental data and is only 2.5+/-0.2 A for the three duplexes that were orientation restrained during the calculation. The global structure simplifies interpretation of multidimensional nuclear Overhauser spectra for high-resolution structure determination. The potential general application of the method for RNA structure determination is discussed.

Chemical-shift-perturbation mapping of the phosphotransfer and catalytic domain interaction in the histidine autokinase CheA from Thermotoga maritima.

Regulating the activity of the histidine autokinase CheA is a central step in bacterial chemotaxis. The CheA autophosphorylation reaction minimally involves two CheA domains, denoted P1 and P4. The kinase domain (P4) binds adenosine triphosphate (ATP) and orients the gamma phosphate for phosphotransfer to a reactive histidine on the phosphoacceptor domain (P1). Three-dimensional triple-resonance experiments allowed sequential assignments of backbone nuclei from P1 and P4 domains as well as the P4 assignments within a larger construct, P3P4, which includes the dimerization domain P3. We have used nuclear magnetic resonance chemical-shift-perturbation mapping to define the interaction of P1 and P3P4 from the hyperthermophile Thermotoga maritima. The observed chemical-shift changes in P1 upon binding suggest that the P1 domain is bound by interactions on the side opposite the histidine that is phosphorylated. The observed shifts in P3P4 upon P1 binding suggest that P1 is bound at a site distinct from the catalytic site on P4. These results argue that the P1 domain is not bound in a mode that leads to productive phosphate transfer from ATP at the catalytic site and imply the presence of multiple binding modes. The binding mode observed may be regulatory or it may reflect the binding mode needed for effective transfer of the histidyl phosphate of P1 to the substrate proteins CheY and CheB. In either case, this work describes the first direct observation of the interaction between P1 and P4 in CheA.