Structure of the cell-binding component of the Clostridium difficile binary toxin reveals a di-heptamer macromolecular assembly.

Targeting Clostridium difficile infection is challenging because treatment options are limited, and high recurrence rates are common. One reason for this is that hypervirulent C. difficile strains often have a binary toxin termed the C. difficile toxin, in addition to the enterotoxins TsdA and TsdB. The C. difficile toxin has an enzymatic component, termed CDTa, and a pore-forming or delivery subunit termed CDTb. CDTb was characterized here using a combination of single-particle cryoelectron microscopy, X-ray crystallography, NMR, and other biophysical methods. In the absence of CDTa, 2 di-heptamer structures for activated CDTb (1.0 MDa) were solved at atomic resolution, including a symmetric (SymCDTb; 3.14 Å) and an asymmetric form (AsymCDTb; 2.84 Å). Roles played by 2 receptor-binding domains of activated CDTb were of particular interest since the receptor-binding domain 1 lacks sequence homology to any other known toxin, and the receptor-binding domain 2 is completely absent in other well-studied heptameric toxins (i.e., anthrax). For AsymCDTb, a Ca2+ binding site was discovered in the first receptor-binding domain that is important for its stability, and the second receptor-binding domain was found to be critical for host cell toxicity and the di-heptamer fold for both forms of activated CDTb. Together, these studies represent a starting point for developing structure-based drug-design strategies to target the most severe strains of C. difficile.

Sulforaphane covalently interacts with the transglutaminase 2 cancer maintenance protein to alter its structure and suppress its activity.

Type 2 transglutaminase (TG2) functions as an important cancer cell survival protein in a range of cancers including epidermal squamous cell carcinoma. TG2 exists in open and closed conformations each of which has a distinct and mutually exclusive activity. The closed conformation has GTP-binding/GTPase activity while the open conformation functions as a transamidase to catalyze protein-protein crosslinking. GTP-binding/GTPase activity is required for TG2 maintenance of the aggressive cancer phenotype. Thus, identifying agents that convert TG2 from the closed to the open GTP-binding/GTPase inactive conformation is an important cancer prevention/treatment strategy. Sulforaphane (SFN) is an important diet-derived cancer prevention agent that is known to possess a reactive isothiocyanate group and has potent anticancer activity. Using a biotin-tagged SFN analog (Biotin-ITC) and kinetic analysis we show that SFN covalently and irreversibly binds to recombinant TG2 to inhibit transamidase activity and shift TG2 to an open/extended conformation, leading to a partial inhibition of GTP binding. We also show that incubation of cancer cells or cancer cell extract with Biotin-ITC results in formation of a TG2/Biotin-ITC complex and that SFN treatment of cancer cells inhibits TG2 transamidase activity and shifts TG2 to an open/extended conformation. These findings identify TG2 as a direct SFN anticancer target in epidermal squamous cell carcinoma.

The Importance of Therapeutically Targeting the Binary Toxin from Clostridioides difficile.

Novel therapeutics are needed to treat pathologies associated with the Clostridioides difficile binary toxin (CDT), particularly when C. difficile infection (CDI) occurs in the elderly or in hospitalized patients having illnesses, in addition to CDI, such as cancer. While therapies are available to block toxicities associated with the large clostridial toxins (TcdA and TcdB) in this nosocomial disease, nothing is available yet to treat toxicities arising from strains of CDI having the binary toxin. Like other binary toxins, the active CDTa catalytic subunit of CDT is delivered into host cells together with an oligomeric assembly of CDTb subunits via host cell receptor-mediated endocytosis. Once CDT arrives in the host cell's cytoplasm, CDTa catalyzes the ADP-ribosylation of G-actin leading to degradation of the cytoskeleton and rapid cell death. Although a detailed molecular mechanism for CDT entry and host cell toxicity is not yet fully established, structural and functional resemblances to other binary toxins are described. Additionally, unique conformational assemblies of individual CDT components are highlighted herein to refine our mechanistic understanding of this deadly toxin as is needed to develop effective new therapeutic strategies for treating some of the most hypervirulent and lethal strains of CDT-containing strains of CDI.

Kröhnke pyridines: Rapid and facile access to Mcl-1 inhibitors.

The tumorigenic activity of upregulated Mcl-1 is manifested by binding the BH3 α-helical death domains of opposing Bcl-2 family members, neutralizing them and preventing apoptosis. Accordingly, the development of Mcl-1 inhibitors largely focuses on synthetic BH3 mimicry. The condensation of α-pyridinium methyl ketone salts and α,ß-unsaturated carbonyl compounds in the presence of a source of ammonia, or the Kröhnke pyridine synthesis, is a simple approach to afford highly functionalized pyridines. We adapted this chemistry to rapidly generate low-micromolar inhibitors of Mcl-1 wherein the 2,4,6-substituents were predicted to mimic the i, i + 2 and i + 7 side chains of the BH3 α-helix.

Mutational studies on resurrected ancestral proteins reveal conservation of site-specific amino acid preferences throughout evolutionary history.

Local protein interactions ("molecular context" effects) dictate amino acid replacements and can be described in terms of site-specific, energetic preferences for any different amino acid. It has been recently debated whether these preferences remain approximately constant during evolution or whether, due to coevolution of sites, they change strongly. Such research highlights an unresolved and fundamental issue with far-reaching implications for phylogenetic analysis and molecular evolution modeling. Here, we take advantage of the recent availability of phenotypically supported laboratory resurrections of Precambrian thioredoxins and ß-lactamases to experimentally address the change of site-specific amino acid preferences over long geological timescales. Extensive mutational analyses support the notion that evolutionary adjustment to a new amino acid may occur, but to a large extent this is insufficient to erase the primitive preference for amino acid replacements. Generally, site-specific amino acid preferences appear to remain conserved throughout evolutionary history despite local sequence divergence. We show such preference conservation to be readily understandable in molecular terms and we provide crystallographic evidence for an intriguing structural-switch mechanism: Energetic preference for an ancestral amino acid in a modern protein can be linked to reorganization upon mutation to the ancestral local structure around the mutated site. Finally, we point out that site-specific preference conservation naturally leads to one plausible evolutionary explanation for the existence of intragenic global suppressor mutations.

Small G proteins Rac1 and Ras regulate serine/threonine protein phosphatase 5 (PP5)·extracellular signal-regulated kinase (ERK) complexes involved in the feedback regulation of Raf1.

Serine/threonine protein phosphatase 5 (PP5, PPP5C) is known to interact with the chaperonin heat shock protein 90 (HSP90) and is involved in the regulation of multiple cellular signaling cascades that control diverse cellular processes, such as cell growth, differentiation, proliferation, motility, and apoptosis. Here, we identify PP5 in stable complexes with extracellular signal-regulated kinases (ERKs). Studies using mutant proteins reveal that the formation of PP5·ERK1 and PP5·ERK2 complexes partially depends on HSP90 binding to PP5 but does not require PP5 or ERK1/2 activity. However, PP5 and ERK activity regulates the phosphorylation state of Raf1 kinase, an upstream activator of ERK signaling. Whereas expression of constitutively active Rac1 promotes the assembly of PP5·ERK1/2 complexes, acute activation of ERK1/2 fails to influence the phosphatase-kinase interaction. Introduction of oncogenic HRas (HRas(V12)) has no effect on PP5-ERK1 binding but selectively decreases the interaction of PP5 with ERK2, in a manner that is independent of PP5 and MAPK/ERK kinase (MEK) activity, yet paradoxically requires ERK2 activity. Additional studies conducted with oncogenic variants of KRas4B reveal that KRas(L61), but not KRas(V12), also decreases the PP5-ERK2 interaction. The expression of wild type HRas or KRas proteins fails to reduce PP5-ERK2 binding, indicating that the effect is specific to HRas(V12) and KRas(L61) gain-of-function mutations. These findings reveal a novel, differential responsiveness of PP5-ERK1 and PP5-ERK2 interactions to select oncogenic Ras variants and also support a role for PP5·ERK complexes in regulating the feedback phosphorylation of PP5-associated Raf1.

Membrane binding and pore formation is Ca 2+ -dependent for the Clostridioides difficile binary toxin.

The C. difficile binary toxin (CDT) enters host cells via endosomal delivery like many other 'AB'-type binary toxins. In this study, the cell-binding component of CDT, termed CDTb, was found to bind and form pores in lipid bilayers upon depleting free Ca 2+ ion concentrations, and not by lowering pH, as found for other binary toxins (i.e., anthrax). Cryoelectron microscopy, nuclear magnetic resonance spectroscopy, surface plasmon resonance, electrochemical impedance spectroscopy, CDT toxicity studies, and site directed mutagenesis show that dissociation of Ca 2+ from a single site in receptor binding domain 1 (RBD1) of CDTb is consistent with a molecular mechanism in which Ca 2+ dissociation from RBD1 induces a "trigger" via conformational exchange that enables CDTb to bind and form pores in endosomal membrane bilayers as free Ca 2+ concentrations decrease during CDT endosomal delivery.

Dendritic Cell-Mediated Cross-Priming by a Bispecific Neutralizing Antibody Boosts Cytotoxic T Cell Responses and Protects Mice against SARS-CoV-2.

Administration of neutralizing antibodies (nAbs) has proved to be effective by providing immediate protection against SARS-CoV-2. However, dual strategies combining virus neutralization and immune response stimulation to enhance specific cytotoxic T cell responses, such as dendritic cell (DC) cross-priming, represent a promising field but have not yet been explored. Here, a broadly nAb, TNT , are first generated by grafting an anti-RBD biparatopic tandem nanobody onto a trimerbody scaffold. Cryo-EM data show that the TNT structure allows simultaneous binding to all six RBD epitopes, demonstrating a high-avidity neutralizing interaction. Then, by C-terminal fusion of an anti-DNGR-1 scFv to TNT , the bispecific trimerbody TNT DNGR-1 is generated to target neutralized virions to type 1 conventional DCs (cDC1s) and promote T cell cross-priming. Therapeutic administration of TNT DNGR-1, but not TNT , protects K18-hACE2 mice from a lethal SARS-CoV-2 infection, boosting virus-specific humoral responses and CD8+ T cell responses. These results further strengthen the central role of interactions with immune cells in the virus-neutralizing antibody activity and demonstrate the therapeutic potential of the Fc-free strategy that can be used advantageously to provide both immediate and long-term protection against SARS-CoV-2 and other viral infections.

Estimating side-chain order in methyl-protonated, perdeuterated proteins via multiple-quantum relaxation violated coherence transfer NMR spectroscopy.

Relaxation violated coherence transfer NMR spectroscopy (Tugarinov et al. in J Am Chem Soc 129:1743-1750, 2007) is an established experimental tool for quantitative estimation of the amplitudes of side-chain motions in methyl-protonated, highly deuterated proteins. Relaxation violated coherence transfer experiments monitor the buildup of methyl proton multiple-quantum coherences that can be created in magnetically equivalent spin-systems as long as their transverse magnetization components relax with substantially different rates. The rate of this build-up is a reporter of the methyl-bearing side-chain mobility. Although the build-up of multiple-quantum (1)H coherences is monitored in these experiments, the decay of the methyl signal during relaxation delays occurs when methyl proton magnetization is in a single-quantum state. We describe a relaxation violated coherence transfer approach where the relaxation of multiple-quantum (1)H-(13)C methyl coherences during the relaxation delay period is quantified. The NMR experiment and the associated fitting procedure that models the time-dependence of the signal build-up, are applicable to the characterization of side-chain order in [(13)CH(3)]-methyl-labeled, highly deuterated protein systems up to ~100 kDa in molecular weight. The feasibility of extracting reliable measures of side-chain order is experimentally verified on methyl-protonated, perdeuterated samples of an 8.5-kDa ubiquitin at 10°C and an 82-kDa Malate Synthase G at 37°C.

Solution NMR evidence for symmetry in functionally or crystallographically asymmetric homodimers.

A recurrent theme of many structural studies of homo-oligomeric protein systems is concerned with verification that the conformation observed in a crystal represents the functionally relevant structure. An asymmetric conformation adopted by two chemically identical subunits in homo-oligomers can represent an intrinsic property of a protein or be an artifact induced by crystal packing forces. Solution NMR studies can distinguish between these two possibilities. Using methyl-based NMR spectroscopy, we provide evidence for symmetry in the absence of ligands in several homodimeric proteins that are either asymmetric functionally and/or adopt different conformations of the two subunits in available X-ray structures.

Simultaneous measurement of ¹H-¹5N and methyl ¹Hm-¹³Cm residual dipolar couplings in large proteins.

A two-dimensional TROSY-based SIM-(13)C(m)-(1)H(m)/(1)H-(15)N NMR experiment for simultaneous measurements of methyl (1) D (CH) and backbone amide (1) D (NH) residual dipolar couplings (RDC) in {U-[(15)N,(2)H]; Ileδ1-[(13)CH(3)]; Leu,Val-[(13)CH(3)/(12)CD(3)]}-labeled samples of large proteins is described. Significant variation in the alignment tensor of the 82-kDa enzyme Malate synthase G is observed as a function of only slight changes in experimental conditions. The SIM-(13)C(m)-(1)H(m)/(1)H-(15)N data sets provide convenient means of establishing the alignment tensor characteristics via the measurement of (1) D (NH) RDCs in the same protein sample.

1HN, 13C, and 15N resonance assignments of the Clostridioides difficile receptor binding domain 2 (CDTb, residues 757-876).

Clostridioides difficile is a bacterial pathogen responsible for the majority of nosocomial infections in the developed world. C. difficile infection (CDI) is difficult to treat in many cases because hypervirulent strains have evolved that contain a third toxin, termed the C. difficile toxin (CDT), in addition to the two enterotoxins TcdA and TcdB. CDT is a binary toxin comprised of an enzymatic, ADP-ribosyltransferase (ART) toxin component, CDTa, and a pore-forming or delivery subunit, CDTb. In the absence of CDTa, CDTb assembles into two distinct di-heptameric states, a symmetric and an asymmetric form with both states having two surface-accessible host cell receptor-binding domains, termed RBD1 and RBD2. RBD1 has a unique amino acid sequence, when aligned to other well-studied binary toxins (i.e., anthrax), and it contains a novel Ca2+-binding site important for CDTb stability. The other receptor binding domain, RBD2, is critically important for CDT toxicity, and a domain such as this is missing altogether in other binary toxins and shows further that CDT is unique when compared to other binary toxins. In this study, the 1H, 13C, and 15N backbone and sidechain resonances of the 120 amino acid RBD2 domain of CDTb (residues 757-876) were assigned sequence-specifically and provide a framework for future NMR-based drug discovery studies directed towards targeting the most virulent strains of CDI.

An Extract of Taro (Colocasia esculenta) Mediates Potent Inhibitory Actions on Metastatic and Cancer Stem Cells by Tumor Cell-Autonomous and Immune-Dependent Mechanisms.

The taro plant, Colocasia esculenta, contains bioactive proteins with potential as cancer therapeutics. Several groups have reported anti-cancer activity in vitro and in vivo of taro-derived extracts (TEs). We reported that TE inhibits metastasis in a syngeneic murine model of Triple-Negative Breast Cancer (TNBC). PURPOSE: We sought to confirm our earlier studies in additional models and to identify novel mechanisms by which efficacy is achieved. METHODS: We employed a panel of murine and human breast and ovarian cancer cell lines to determine the effect of TE on tumor cell viability, migration, and the ability to support cancer stem cells. Two syngeneic models of TNBC were employed to confirm our earlier report that TE potently inhibits metastasis. Cancer stem cell assays were employed to determine the ability of TE to inhibit tumorsphere-forming ability and to inhibit aldehyde dehydrogenase activity. To determine if host immunity contributes to the mechanism of metastasis inhibition, efficacy was assessed in immune-compromised mice. RESULTS: We demonstrate that viability of some, but not all cell lines is inhibited by TE. Likewise, tumor cell migration is inhibited by TE. Using 2 immune competent, syngeneic models of TNBC, we confirm our earlier findings that tumor metastasis is potently inhibited by TE. We also demonstrate, for the first time, that TE directly inhibits breast cancer stem cells. Administration of TE to mice elicits expansion of several spleen cell populations but it was not known if host immune cells contribute to the mechanism by which TE inhibits tumor cell dissemination. In novel findings, we now show that the ability of TE to inhibit metastasis relies on immune T-cell-dependent, but not B cell or Natural Killer (NK)-cell-dependent mechanisms. Thus, both tumor cell-autonomous and host immune factors contribute to the mechanisms underlying TE efficacy. Our long-term goal is to evaluate TE efficacy in clinical trials. Most of our past studies as well as many of the results reported in this report were carried out using an isolation protocol described earlier (TE). In preparation for a near future clinical trial, we have now developed a strategy to isolate an enriched taro fraction, TE-method 2, (TE-M2) as well as a more purified subfraction (TE-M2F1) which can be scaled up under Good Manufacturing Practice (GMP) conditions for evaluation in human subjects. We demonstrate that TE-M2 and TE-M2F1 retain the anti-metastatic properties of TE. CONCLUSIONS: These studies provide further support for the continued examination of biologically active components of Colocasia esculenta as potential new therapeutic entities and identify a method to isolate sufficient quantities under GMP conditions to conduct early phase clinical studies.

Physiologically Relevant Free Ca2+ Ion Concentrations Regulate STRA6-Calmodulin Complex Formation via the BP2 Region of STRA6.

The interaction of calmodulin (CaM) with the receptor for retinol uptake, STRA6, involves an α-helix termed BP2 that is located on the intracellular side of this homodimeric transporter (Chen et al., 2016 [1]). In the absence of Ca2+, NMR data showed that a peptide derived from BP2 bound to the C-terminal lobe (C-lobe) of Mg2+-bound CaM (MgCaM). Upon titration of Ca2+ into MgCaM-BP2, NMR chemical shift perturbations (CSPs) were observed for residues in the C-lobe, including those in the EF-hand Ca2+-binding domains, EF3 and EF4 (CaKD = 60 ± 7 nM). As higher concentrations of free Ca2+ were achieved, CSPs occurred for residues in the N-terminal lobe (N-lobe) including those in EF1 and EF2 (CaKD = 1000 ± 160 nM). Thermodynamic and kinetic Ca2+ binding studies showed that BP2 addition increased the Ca2+-binding affinity of CaM and slowed its Ca2+ dissociation rates (koff) in both the C- and N-lobe EF-hand domains, respectively. These data are consistent with BP2 binding to the C-lobe of CaM at low free Ca2+ concentrations (<100 nM) like those found at resting intracellular levels. As free Ca2+ levels approach 1000 nM, which is typical inside a cell upon an intracellular Ca2+-signaling event, BP2 is shown here to interact with both the N- and C-lobes of Ca2+-loaded CaM (CaCaM-BP2). Because this structural rearrangement observed for the CaCaM-BP2 complex occurs as intracellular free Ca2+ concentrations approach those typical of a Ca2+-signaling event (CaKD = 1000 ± 160 nM), this conformational change could be relevant to vitamin A transport by full-length CaCaM-STRA6.

Engineering subtilisin proteases that specifically degrade active RAS.

We describe the design, kinetic properties, and structures of engineered subtilisin proteases that degrade the active form of RAS by cleaving a conserved sequence in switch 2. RAS is a signaling protein that, when mutated, drives a third of human cancers. To generate high specificity for the RAS target sequence, the active site was modified to be dependent on a cofactor (imidazole or nitrite) and protease sub-sites were engineered to create a linkage between substrate and cofactor binding. Selective proteolysis of active RAS arises from a 2-step process wherein sub-site interactions promote productive binding of the cofactor, enabling cleavage. Proteases engineered in this way specifically cleave active RAS in vitro, deplete the level of RAS in a bacterial reporter system, and also degrade RAS in human cell culture. Although these proteases target active RAS, the underlying design principles are fundamental and will be adaptable to many target proteins.

Alanine methyl groups as NMR probes of molecular structure and dynamics in high-molecular-weight proteins.

The importance and utility of Ala(ß) methyl groups as NMR probes of molecular structure and dynamics in high-molecular-weight proteins is explored. Using (2)H and (13)C relaxation measurements in {U-(2)H; Ala(ß)-[(13)CHD(2)]}-labeled Malate Synthase G (MSG)--an 82-kDa monomeric enzyme that contains 73 Ala(ß) methyl groups--we show that the vast majority of selectively labeled Ala(ß) methyls are highly ordered. A number of NMR applications used for solution studies of structure and dynamics of large protein molecules can benefit from proximity of Ala(ß) methyls to the protein backbone and their high degree of ordering. In the case of MSG, these applications include the measurement of (1)H-(13)C residual dipolar couplings in Ala(ß) methyls, characterization of slow (µs-to-ms) dynamics at the substrates' binding sites, and methyl-TROSY-based NOE spectroscopy performed on {U-(2)H; Ala(ß)-[(13)CH(3)]; Ile(δ1)-[(13)CH(3)]; Leu,Val-[(13)CH(3)/(12)CD(3)]}-labeled samples where the number of methyl probes for derivation of distance restraints is maximized compared to the state-of-the-art ILV labeling methodology.

High resolution measurement of methyl 13C(m)-13C and 1H(m)-13C(m) residual dipolar couplings in large proteins.

NMR methodology is developed for high-resolution, accurate measurements of methyl (1)H(m)-(13)C(m) ((1)D(CH)) and (13)C(m)-(13)C ((1)D(CC)) residual dipolar couplings (RDCs) in ILV-methyl-protonated high-molecular-weight proteins. Both types of RDCs are measured in a three-dimensional (3D) mode that allows dispersion of correlations to the third ((13)C(ß/γ)) dimension, alleviating the problem of overlap of methyl resonances in highly complex and methyl-abundant protein structures. The methodology is applied to selectively ILV-protonated 82-kDa monomeric enzyme malate synthase G (MSG) that contains 273 ILV methyl groups with substantial overlap of methyl resonances in 2D methyl (1)H-(13)C correlation maps. A good agreement is observed between the measured RDCs of both types and those calculated from the crystallographic coordinates of MSG for the residues with low-amplitude internal dynamics. Although the measurement of (1)D(CH) RDCs from the acquisition dimension of NMR spectra imposes certain limitations on the accuracy of obtained (1)D(CH) values, (1)D(CH) couplings can be approximately corrected for cross-correlated relaxation effects. The ratios of (1)D(CH) and (1)D(CC) couplings ((1)D(CH)/(1)D(CC)) are independent of methyl axis dynamics and the details of residual alignment [Ottiger, M.; Bax, A. J. Am. Chem. Soc. 1999, 121, 4690.]. The (1)D(CH)/(1)D(CC) ratios obtained in MSG can therefore validate the employed correction scheme.

Variation in quadrupole couplings of alpha deuterons in ubiquitin suggests the presence of C(alpha)-H(alpha)...O=C hydrogen bonds.

Nuclear quadrupolar couplings are sensitive probes of hydrogen bonding. Experimental quadrupolar coupling constants of alpha deuterons (D(alpha) QCC) are reported for the residues of human ubiquitin that do not experience large-amplitude internal dynamics on the pico- to nanosecond time scale. Two different methods for D(alpha) QCC estimation are employed: (i) direct estimation of D(alpha) QCC values from R(1) and R(2) (2)H D(alpha) rates using the dynamics parameters (S(C(alpha)-H(alpha))(2)) derived from 1 micros molecular dynamics simulations as well as from (13)C(alpha) relaxation measurements and (ii) indirect measurements via scalar relaxation of the second kind that affects (13)C(alpha) relaxation rates in (13)C(alpha)-D(alpha) spin systems. A relatively large variability of D(alpha) QCC values is produced by both methods. The average value of 170.6 +/- 3 kHz is derived from the combined data set, with D(alpha) QCC values ranging from 159.2 to 177.2 kHz. The set of lowest quadrupolar couplings in all data sets corresponds to the residues that are likely to form weak C(alpha)-H(alpha)...O=C hydrogen bonds as predicted from the analysis of short H(alpha)...O distances in three-dimensional structures of ubiquitin. These D(alpha) nuclei show up to 10 kHz reduction in their QCC values, which is in agreement with earlier solid-state NMR measurements in alpha deuterons of glycine. A statistically significant correlation is observed between the QCC values of alpha-deuterons and the inverse cube of C(alpha)-H(alpha)...O=C distances in ubiquitin.

Correction to: 1HN, 13C, and 15N resonance assignments of human calmodulin bound to a peptide derived from the STRA6 vitamin A transporter (CaMBP2).

The article 1HN, 13C, and 15N resonance assignments of human calmodulin bound to a peptide derived from the STRA6 vitamin A transporter (CaMBP2), written by Kristen M. Varney, Paul T. Wilder, Raquel Godoy-Ruiz, Filippo Mancia and David J. Weber, was originally published Online First without Open Access. After publication in volume 13, issue 2, page [275-278] the author decided to opt for Open Choice and to make the article an Open Access publication.

1HN, 13C, and 15N resonance assignments of human calmodulin bound to a peptide derived from the STRA6 vitamin A transporter (CaMBP2).

Vitamin A is a necessary nutrient for all mammals, and it is required for the transcription of many genes and vital for vision. While fasting, the vitamin A alcohol form (Retinol) from storage in the liver is mobilized and transported through the bloodstream while bound to retinol binding protein (RBP). Details of how exactly vitamin A is released from RBP and taken into the cells are still unclear. As part of the effort to elucidate the specifics of this process, single-particle cryo-electron microscopy structural studies of STRA6 (the RBP receptor 75-kDa transmembrane receptor protein) were recently reported by Chen et al. (Science, https://doi.org/10.1126/science.aad8266 , 2016). Interestingly, STRA6 from zebrafish was shown to be a stable dimer and bound to calmodulin (CaM), forming a 180-kDa complex. The topology of the STRA6 complex includes 18 transmembrane helices (nine per protomer) and two long horizontal intramembrane helices interacting at the dimer core (Chen et al., in Science, https://doi.org/10.1126/science.aad8266 , 2016). CaM was shown to interact with three regions of STRA6, termed CaMBP1, CaMBP2, and CaMBP3, with the most extensive interactions involving CaMBP2. To further our understanding of Ca2+-dependence of CaM-STRA6 complex formation, studies of the structure and dynamic properties of the CaMBP2-CaM complex were initiated. For this, the 1HN, 13C, and 15N backbone resonance assignments of the 148 amino acid Ca2+-bound calmodulin protein bound to the 27-residue CaMBP2 peptide derived from STRA6 were completed here using heteronuclear multidimensional NMR spectroscopy.