Structural insights into auxiliary cofactor usage by radical S-adenosylmethionine enzymes

Radical S-adenosylmethionine (SAM) enzymes use a common catalytic core for diverse transformations. While all radical SAM enzymes bind a Fe₄S₄ cluster via a characteristic tri-cysteine motif, many bind additional metal cofactors. Recently reported structures of radical SAM enzymes that use methylcobalamin or additional iron-sulfur clusters as cosubstrates show that these auxiliary units are anchored by N- and C-terminal domains that vary significantly in size and topology. Despite this architectural diversity, all use a common surface for auxiliary cofactor docking. In the sulfur insertion and metallocofactor assembly systems evaluated here, interaction with iron-sulfur cluster assembly proteins or downstream scaffold proteins is an important component of catalysis. Structures of these complexes represent important new frontiers in structural analysis of radical SAM enzymes.     

A new method for extrusion of iron-sulfur cores from active centers of proteins

A new method for identification and quantitation of [2Fe-2S] and [4Fe-4S] types of iron-sulfur centers in proteins is presented. The method relies on the solubilization of C₆H₅SH, [Fe₂S₂(SC₆H₅)₄]^?2 and [Fe₄S₄(SC₆H₅)₄]^?2 in aqueous solutions containing 5–17 vol % N,N-dimethylformamide by 5 vol % Triton X-100. Quantitative removal of the Fe₂S₂ core of Spinach ferredoxin is achieved in media containing 80 vol % water. Advantages over previous core extrusion methods include avoidance of toxic hexamethylphosphoramide, a smaller percentage of organic solvent, increased sensitivity and (for Spinach ferredoxin) decreased extrusion time.     

M?ssbauer spectroscopy of the nitrogenase proteins from Klebsiella pneumoniae. Structural assignments and mechanistic conclusions

The Mo–Fe protein and the Fe protein which together constitute the nitrogenase of Klebsiella pneumoniae were prepared from bacteria grown in ⁵⁷Fe-enriched medium. The Mössbauer spectrum of the Mo–Fe protein, as isolated in the presence of Na₂S₂O₄, showed that the protein contained three iron species, called M4, M5 and M6. The area of the spectrum associated with species M4, with δ=0.65mm/s and ΔE=3.05mm/s at 4.2°K, corresponded to two iron atoms/molecule of protein and it is interpreted as being due to a high-spin ferrous, spin-coupled pair of iron atoms. The iron atoms of species M4 may be involved in the quaternary structure of the protein. Species M5, with δ=0.61mm/s and ΔE=0.83mm/s at 77°K, corresponded to eight iron atoms/molecule of protein and is interpreted as being due to Fe₄S₄ or Fe₂S₂ low-spin ferrous iron clusters. Species M6, with δ=0.37mm/s and ΔE=0.71mm/s at 77°K, also corresponded to eight iron atoms/molecule of protein and, at 4.2°K, became a broad shallow absorption, characteristic of magnetic hyperfine interaction. Oxidation of the Mo–Fe protein with the redox dye Lauth''s Violet did not affect the activity of the protein but changed species M4, M5 and M6 into the species M1 (δ=0.37mm/s, ΔE=0.75mm/s at 77°K, broad magnetic component at 4.2°K) and M2 (δ=0.35mm/s, ΔE=0.9mm/s at 4.2°K). In the presence of the Fe protein, Na₂S₂O₄, ATP and Mg²⁺, the M6 component of the Mo–Fe protein was replaced by species M7 with δ=0.46mm/s, ΔE=1.04mm/s at 4.2°K. The change in Mössbauer parameters associated with the M6 → M7 transformation was very similar to the change observed on reduction of the high-potential Fe protein from Chromatium vinosum. In contrast, Na₂S₂O₄-reduced Fe protein contained only one type of iron cluster (F4). Species F4 had δ=0.50mm/s, ΔE=0.9mm/s at 195°K, and at 4.2°K broadened in a manner characteristic of a magnetic hyperfine interaction, associated with half-integral spin, equally distributed over all four atoms of the Fe protein. The Mössbauer spectra of the Mo–Fe and the Fe protein under argon were unaffected by the reducible substrates N₂ and C₂H₂ and the inhibitor CO in the presence of ATP, Mg²⁺ and Na₂S₂O₄. A number of Mössbauer spectral species associated with inactivated Mo–Fe and Fe proteins are described and discussed.     

Functional mutation of multiple solvent-exposed loops in the Ecballium elaterium trypsin inhibitor-II cystine knot miniprotein

Background

The Ecballium elaterium trypsin inhibitor (EETI-II), a 28-amino acid member of the knottin family of peptides, contains three interwoven disulfide bonds that form multiple solvent-exposed loops. Previously, the trypsin binding loop of EETI-II has been engineered to confer binding to several alternative molecular targets. Here, EETI-II was further explored as a molecular scaffold for polypeptide engineering by evaluating the ability to mutate two of its structurally adjacent loops.

Methodology/Principal Findings

Yeast surface display was used to engineer an EETI-II mutant containing two separate integrin binding epitopes. The resulting knottin peptide was comprised of 38 amino acids, and contained 11- and 10-residue loops compared to wild-type EETI-II, which naturally contains 6- and 5-residue loops, respectively. This knottin peptide bound to α_vβ₃ and α_vβ₅ integrins with affinities in the low nanomolar range, but bound weakly to the related integrins α₅β₁ and α_iibβ₃. In addition, the engineered knottin peptide inhibited tumor cell adhesion to vitronectin, an extracellular matrix protein that binds to α_vβ₃ and α_vβ₅ integrins. A ⁶⁴Cu radiolabeled version of this knottin peptide demonstrated moderate serum stability and excellent tumor-to-muscle and tumor-to-blood ratios by positron emission tomography imaging in human tumor xenograft models. Tumor uptake was ∼3–5% injected dose per gram (%ID/g) at one hour post injection, with rapid clearance of probe through the kidneys.

Conclusions/Significance

We demonstrated that multiple loops of EETI-II can be mutated to bind with high affinity to tumor-associated integrin receptors. The resulting knottin peptide contained 21 (>50%) non-native amino acids within two mutated loops, indicating that extended loop lengths and sequence diversity were well tolerated within the EETI-II scaffold. A radiolabeled version of this knottin peptide showed promise for non-invasive imaging of integrin expression in living subjects. However, reduced serum and metabolic stability were observed compared to an engineered integrin-binding EETI-II knottin peptide containing only one mutated loop.     

Heterometallic [AgFe3S4] ferredoxin variants: synthesis, characterization, and the first crystal structure of an engineered heterometallic iron–sulfur protein

Heterometallic [AgFe₃S₄] iron–sulfur clusters assembled in wild-type Pyrococcus furiosus ferredoxin and two variants, D14C and D14H, are characterized. The crystal structure of the [AgFe₃S₄] D14C variant shows that the silver(I) ion is indeed part of the cluster and is coordinated to the thiolate group of residue 14. Cyclic voltammetry shows one redox pair with a reduction potential of +220 mV versus the standard hydrogen electrode which is assigned to the [AgFe₃S₄]^2+/+ couple. The oxidized form of the [AgFe₃S₄] D14C variant is stable in the presence of dioxygen, whereas the oxidized forms of the [AgFe₃S₄] wild type and D14H variants convert to the [Fe₃S₄] ferredoxin form. The monovalent d ¹⁰ silver(I) ion stabilizes the [Fe₃S₄]^+/0 cluster fragment, as opposed to divalent d ¹⁰ metal ions, resulting in more than 0.4 V difference in reduction potentials between the silver(I) and, e.g., zinc(II) heterometallic [MFe₃S₄] ferredoxins. The trend in reduction potentials for the variants containing the [AgFe₃S₄] cluster is wild type ≤ D14C < D14H and shows the same trend as reported for the variants containing the [Fe₃S₄] cluster, but is different from the D14C < D14H < wild type trend reported for the [Fe₄S₄] ferredoxin. The similarity in the reduction potential trend for the variants containing the heterometallic [AgFe₃S₄] cluster and the [Fe₃S₄] cluster can be rationalized in terms of the electrostatic influence of the residue 14 side chains, rather than the dissociation constant of this residue, as is the case for [Fe₄S₄] ferredoxins. The trends in reduction potentials are in line with there being no electronic coupling between the silver(I) ion and the Fe₃S₄ fragment.     

Parsing redox potentials of five ferredoxins found within Thermotoga maritima

Most organisms contain multiple soluble protein‐based redox carriers such as members of the ferredoxin (Fd) family, that contain one or more iron–sulfur clusters. The potential redundancy of Fd proteins is poorly understood, particularly in connection to the ability of Fd proteins to deliver reducing equivalents to members of the “radical SAM,” or S‐adenosylmethionine radical enzyme (ARE) superfamily, where the activity of all known AREs requires that an essential iron–sulfur cluster bound by the enzyme be reduced to the catalytically relevant [Fe₄S₄]¹⁺ oxidation state. As it is still unclear whether a single Fd in a given organism is specific to individual redox partners, we have examined the five Fd proteins found within Thermotoga maritima via direct electrochemistry, to compare them in a side‐by‐side fashion for the first time. While a single [Fe₄S₄]‐cluster bearing Fd (TM0927) has a potential of ?420 mV, the other four 2x[Fe₄S₄]‐bearing Fds (TM1175, TM1289, TM1533, and TM1815) have potentials that vary significantly, including cases where the two clusters of the same Fd are essentially coincident (e.g., TM1175) and those where the potentials are well separate (TM1815).     

De novo design of a non-natural fold for an iron-sulfur protein: Alpha-helical coiled-coil with a four-iron four-sulfur cluster binding site in its central core

Using a ‘metal-first’ approach, we computationally designed, prepared, and characterized a four-iron four-sulfur (Fe₄S₄) cluster protein with a non-natural α-helical coiled-coil fold. The novelty of this fold lies in the placement of a Fe₄S₄ cluster within the hydrophobic core of a four-helix bundle, making it unique among previous iron-sulfur (FeS) protein designs, and different from known natural FeS proteins. The apoprotein, recombinantly expressed and purified from E. coli, readily self-assembles with Fe₄S₄ clusters in vitro. UV-Vis absorption and CD spectroscopy, elemental analysis, gel filtration, and analytical ultracentrifugation confirm that the protein is folded and assembled as designed, namely, α-helical coiled-coil binding a single Fe₄S₄ cluster. Dithionite-reduced holoprotein samples have characteristic rhombic EPR spectra, typical of low-potential, [Fe₄S₄]⁺ (S = 1/2), with g values of g_z,y = (1.970, 1.975), and g_x = 2.053. The temperature, and power dependence of the signal intensity were also characteristic of [Fe₄S₄]⁺ clusters with very efficient spin relaxation, but almost without any interaction between adjacent clusters. The new design is very promising although optimization is required, particularly for preventing aggregation, and adding second shell interactions to stabilize the reduced state. Its main advantage is its extendibility into a multi-FeS cluster protein by simply duplicating and translating the binding site along the coiled-coil axis. This opens new possibilities for designing protein-embedded redox chains that may be used as “wires” for coupling any given set of redox enzymes.     

Structure and electrochemistry of proteins harboring iron-sulfur clusters of different nuclearities. Part IV. Canonical,non-canonical and hybrid iron-sulfur proteins

A plethora of proteins are able to express iron-sulfur clusters, but have a clear picture of the different types of proteins and the different iron-sulfur clusters they harbor it is not easy.In the last five years we have reviewed structure/electrochemistry of metalloproteins expressing: (i) single types of iron-sulfur clusters (namely: {Fe(Cys)₄}, {[Fe₂S₂](Cys)₄}, {[Fe₂S₂](Cys)₃(X)} (X?=?Asp, Arg, His), {[Fe₂S₂](Cys)₂(His)₂}, {[Fe₃S₄](Cys)₃}, {[Fe₄S₄](Cys)₄} and {[Fe₄S₄](Cys)₃(nonthiolate ligand)} cores); (ii) metalloproteins harboring iron-sulfur centres of different nuclearities (namely: [4Fe-4S] and [2Fe-2S], [4Fe-4S] and [3Fe-4S], and [4Fe-4S], [3Fe-4S] and [2Fe-2S] clusters. Our target is now to review structure and electrochemistry of proteins harboring canonical, non-canonical and hybrid iron-sulfur proteins.     

FXYD proteins stabilize Na,K-ATPase: amplification of specific phosphatidylserine-protein interactions

FXYD proteins are a family of seven small regulatory proteins, expressed in a tissue-specific manner, that associate with Na,K-ATPase as subsidiary subunits and modulate kinetic properties. This study describes an additional property of FXYD proteins as stabilizers of Na,K-ATPase. FXYD1 (phospholemman), FXYD2 (γ subunit), and FXYD4 (CHIF) have been expressed in Escherichia coli and purified. These FXYD proteins associate spontaneously in vitro with detergent-soluble purified recombinant human Na,K-ATPase (α1β1) to form α1β1FXYD complexes. Compared with the control (α1β1), all three FXYD proteins strongly protect Na,K-ATPase activity against inactivation by heating or excess detergent (C₁₂E₈), with effectiveness FXYD1 > FXYD2 ≥ FXYD4. Heating also inactivates E₁ ↔ E₂ conformational changes and cation occlusion, and FXYD1 protects strongly. Incubation of α1β1 or α1β1FXYD complexes with guanidinium chloride (up to 6 m) causes protein unfolding, detected by changes in protein fluorescence, but FXYD proteins do not protect. Thus, general protein denaturation is not the cause of thermally mediated or detergent-mediated inactivation. By contrast, the experiments show that displacement of specifically bound phosphatidylserine is the primary cause of thermally mediated or detergent-mediated inactivation, and FXYD proteins stabilize phosphatidylserine-Na,K-ATPase interactions. Phosphatidylserine probably binds near trans-membrane segments M9 of the α subunit and the FXYD protein, which are in proximity. FXYD1, FXYD2, and FXYD4 co-expressed in HeLa cells with rat α1 protect strongly against thermal inactivation. Stabilization of Na,K-ATPase by three FXYD proteins in a mammalian cell membrane, as well the purified recombinant Na,K-ATPase, suggests that stabilization is a general property of FXYD proteins, consistent with a significant biological function.     

Calcium Currents Are Enhanced by ��2��-1 Lacking Its Membrane Anchor

The accessory α₂δ subunits of voltage-gated calcium channels are membrane-anchored proteins, which are highly glycosylated, possess multiple disulfide bonds, and are post-translationally cleaved into α₂ and δ. All α₂δ subunits have a C-terminal hydrophobic, potentially trans-membrane domain and were described as type I transmembrane proteins, but we found evidence that they can be glycosylphosphatidylinositol-anchored. To probe further the function of membrane anchoring in α₂δ subunits, we have now examined the properties of α₂δ-1 constructs truncated at their putative glycosylphosphatidylinositol anchor site, located before the C-terminal hydrophobic domain (α₂δ-1ΔC-term). We find that the majority of α₂δ-1ΔC-term is soluble and secreted into the medium, but unexpectedly, some of the protein remains associated with detergent-resistant membranes, also termed lipid rafts, and is extrinsically bound to the plasma membrane. Furthermore, heterologous co-expression of α₂δ-1ΔC-term with Ca_V2.1/β1b results in a substantial enhancement of the calcium channel currents, albeit less than that produced by wild-type α₂δ-1. These results call into question the role of membrane anchoring of α₂δ subunits for calcium current enhancement.     

The Streptococcal Collagen-binding Protein CNE Specifically Interferes with ��V��3-mediated Cellular Interactions with Triple Helical Collagen

Collagen fibers expose distinct domains allowing for specific interactions with other extracellular matrix proteins and cells. To investigate putative collagen domains that govern integrin α_Vβ₃-mediated cellular interactions with native collagen fibers we took advantage of the streptococcal protein CNE that bound native fibrillar collagens. CNE specifically inhibited α_Vβ₃-dependent cell-mediated collagen gel contraction, PDGF BB-induced and α_Vβ₃-mediated adhesion of cells, and binding of fibronectin to native collagen. Using a Toolkit composed of overlapping, 27-residue triple helical segments of collagen type II, two CNE-binding sites present in peptides II-1 and II-44 were identified. These peptides lack the major binding site for collagen-binding β₁ integrins, defined by the peptide GFOGER. Peptide II-44 corresponds to a region of collagen known to bind collagenases, discoidin domain receptor 2, SPARC (osteonectin), and fibronectin. In addition to binding fibronectin, peptide II-44 but not II-1 inhibited α_Vβ₃-mediated collagen gel contraction and, when immobilized on plastic, supported adhesion of cells. Reduction of fibronectin expression by siRNA reduced PDGF BB-induced α_Vβ₃-mediated contraction. Reconstitution of collagen types I and II gels in the presence of CNE reduced collagen fibril diameters and fibril melting temperatures. Our data indicate that contraction proceeded through an indirect mechanism involving binding of cell-produced fibronectin to the collagen fibers. Furthermore, our data show that cell-mediated collagen gel contraction does not directly depend on the process of fibril formation.     

Alzheimer's disease drug candidates stabilize A-β protein native structure by interacting with the hydrophobic core

Deposition of amyloid fibrils, consisting primarily of Aβ₄₀ and Aβ₄₂ peptides, in the extracellular space in the brain is a major characteristic of Alzheimer''s disease (AD). We recently developed new (to our knowledge) drug candidates for AD that inhibit the fibril formation of Aβ peptides and eliminate their neurotoxicity. We performed all-atom molecular-dynamics simulations on the Aβ₄₂ monomer at its α-helical conformation and a pentamer fibril fragment of Aβ₄₂ peptide with or without LRL and fluorene series compounds to investigate the mechanism of inhibition. The results show that the active drug candidates, LRL22 (EC₅₀ = 0.734 μM) and K162 (EC₅₀ = 0.080 μM), stabilize hydrophobic core I of Aβ₄₂ peptide (residues 17–21) to its α-helical conformation by interacting specifically in this region. The nonactive drug candidates, LRL27 (EC₅₀ > 10 μM) and K182 (EC₅₀ > 5 μM), have little to no similar effect. This explains the different behavior of the drug candidates in experiments. Of more importance, this phenomenon indicates that hydrophobic core I of the Aβ₄₂ peptide plays a major mechanistic role in the formation of amyloid fibrils, and paves the way for the development of new drugs against AD.     

Suboptimal Activation of Protease-activated Receptors Enhances ��2��1 Integrin-mediated Platelet Adhesion to Collagen

Thrombin and fibrillar collagen are potent activators of platelets at sites of vascular injury. Both agonists cause platelet shape change, granule secretion, and aggregation to form the primary hemostatic plug. Human platelets express two thrombin receptors, protease-activated receptors 1 and 4 (PAR1 and PAR4) and two collagen receptors, the α₂β₁ integrin (α₂β₁) and the glycoprotein VI (GPVI)/FcRγ chain complex. Although these receptors and their signaling mechanisms have been intensely studied, it is not known whether and how these receptors cooperate in the hemostatic function of platelets. This study examined cooperation between the thrombin and collagen receptors in platelet adhesion by utilizing a collagen-related peptide (α2-CRP) containing the α₂β₁-specific binding motif, GFOGER, in conjunction with PAR-activating peptides. We demonstrate that platelet adhesion to α2-CRP is substantially enhanced by suboptimal PAR activation (agonist concentrations that do not stimulate platelet aggregation) using the PAR4 agonist peptide and thrombin. The enhanced adhesion induced by suboptimal PAR4 activation was α₂β₁-dependent and GPVI/FcRγ-independent as revealed in experiments with α₂β₁- or FcRγ-deficient mouse platelets. We further show that suboptimal activation of other platelet G_q-linked G protein-coupled receptors (GPCRs) produces enhanced platelet adhesion to α2-CRP. The enhanced α₂β₁-mediated platelet adhesion is controlled by phospholipase C (PLC), but is not dependent on granule secretion, activation of α_IIbβ₃ integrin, or on phosphoinositol-3 kinase (PI3K) activity. In conclusion, we demonstrate a platelet priming mechanism initiated by suboptimal activation of PAR4 or other platelet G_q-linked GPCRs through a PLC-dependent signaling cascade that promotes enhanced α₂β₁ binding to collagens containing GFOGER sites.     

Expression,purification and characterization of an iron-sulfur cluster assembly protein,IscU, from <Emphasis Type="Italic">Acidithiobacillus ferrooxidans</Emphasis>

An iron-sulfur cluster assembly protein, IscU, is encoded by the operon iscSUA in Acidithiobacillus ferrooxidans. The gene of IscU was cloned and expressed in Escherichia coli. The protein was purified by one-step affinity chromatography to homogeneity. The protein was in apo-form, the [Fe₂S₂] cluster could be assembled in apoIscU with Fe²⁺ and sulfide in vitro, and in the presence of IscA and IscS, the IscU could utilize l-cysteine and Fe²⁺ to synthesize [Fe₂S₂] cluster in the protein. Site-directed mutagenesis for the protein revealed that Cys37, Asp39, Cys63 and Cys106 were involved in ligating with the [Fe₂S₂] cluster.     

Translation, stability, and resistance to decapping of mRNAs containing caps substituted in the triphosphate chain with BH3, Se, and NH

Decapping is an essential step in multiple pathways of mRNA degradation. Previously, we synthesized mRNAs containing caps that were resistant to decapping, both to dissect the various pathways for mRNA degradation and to stabilize mRNA for more sustained protein expression. mRNAs containing an α-β CH₂ group are resistant to in vitro cleavage by the decapping enzyme hDcp2 but poorly translated. mRNAs containing an S substitution at the β-phosphate are well translated but only partially resistant to hDcp2. We now describe seven new cap analogs substituted at the β-phosphate with BH₃ or Se, or substituted at either the α-β or β-γ O with NH. The analogs differ in affinity for eIF4E and efficiency of in vitro incorporation into mRNA by T7 RNA polymerase. Luciferase mRNAs capped with these analogs differ in resistance to hDcp2 hydrolysis in vitro, translational efficiency in rabbit reticulocyte lysate and in HeLa cells, and stability in HeLa cells. Whereas mRNAs capped with m₂^7,2′-OGpp_SpG were previously found to have the most favorable properties of translational efficiency and stability in mammalian cells, mRNAs capped with m⁷Gpp_BH3pm⁷G are translated with the same efficiency but are more stable. Interestingly, some mRNAs exhibit a lag of up to 60 min before undergoing first-order decay (t_1/2 ≅ 25 min). Only mRNAs that are efficiently capped, resistant to decapping in vitro, and actively translated have long lag phases.     

Proper Restoration of Excitation-Contraction Coupling in the Dihydropyridine Receptor ��1-null Zebrafish Relaxed Is an Exclusive Function of the ��1a Subunit

The paralyzed zebrafish strain relaxed carries a null mutation for the skeletal muscle dihydropyridine receptor (DHPR) β_1a subunit. Lack of β_1a results in (i) reduced membrane expression of the pore forming DHPR α_1S subunit, (ii) elimination of α_1S charge movement, and (iii) impediment of arrangement of the DHPRs in groups of four (tetrads) opposing the ryanodine receptor (RyR1), a structural prerequisite for skeletal muscle-type excitation-contraction (EC) coupling. In this study we used relaxed larvae and isolated myotubes as expression systems to discriminate specific functions of β_1a from rather general functions of β isoforms. Zebrafish and mammalian β_1a subunits quantitatively restored α_1S triad targeting and charge movement as well as intracellular Ca²⁺ release, allowed arrangement of DHPRs in tetrads, and most strikingly recovered a fully motile phenotype in relaxed larvae. Interestingly, the cardiac/neuronal β_2a as the phylogenetically closest, and the ancestral housefly β_M as the most distant isoform to β_1a also completely recovered α_1S triad expression and charge movement. However, both revealed drastically impaired intracellular Ca²⁺ transients and very limited tetrad formation compared with β_1a. Consequently, larval motility was either only partially restored (β_2a-injected larvae) or not restored at all (β_M). Thus, our results indicate that triad expression and facilitation of 1,4-dihydropyridine receptor (DHPR) charge movement are common features of all tested β subunits, whereas the efficient arrangement of DHPRs in tetrads and thus intact DHPR-RyR1 coupling is only promoted by the β_1a isoform. Consequently, we postulate a model that presents β_1a as an allosteric modifier of α_1S conformation enabling skeletal muscle-type EC coupling.Excitation-contraction (EC)³ coupling in skeletal muscle is critically dependent on the close interaction of two distinct Ca²⁺ channels. Membrane depolarizations of the myotube are sensed by the voltage-dependent 1,4-dihydropyridine receptor (DHPR) in the sarcolemma, leading to a rearrangement of charged amino acids (charge movement) in the transmembrane segments S4 of the pore-forming DHPR α_1S subunit (1, 2). This conformational change induces via protein-protein interaction (3, 4) the opening of the sarcoplasmic type-1 ryanodine receptor (RyR1) without need of Ca²⁺ influx through the DHPR (5). The release of Ca²⁺ from the sarcoplasmic reticulum via RyR1 consequently induces muscle contraction. The protein-protein interaction mechanism between DHPR and RyR1 requires correct ultrastructural targeting of both channels. In Ca²⁺ release units (triads and peripheral couplings) of the skeletal muscle, groups of four DHPRs (tetrads) are coupled to every other RyR1 and hence are geometrically arranged following the RyR-specific orthogonal arrays (6).The skeletal muscle DHPR is a heteromultimeric protein complex, composed of the voltage-sensing and pore-forming α_1S subunit and auxiliary subunits β_1a, α₂δ-1, and γ₁ (7). While gene knock-out of the DHPR γ₁ subunit (8, 9) and small interfering RNA knockdown of the DHPR α₂δ-1 subunit (10-12) have indicated that neither subunit is essential for coupling of the DHPR with RyR1, the lack of the α_1S or of the intracellular β_1a subunit is incompatible with EC coupling and accordingly null model mice die perinatally due to asphyxia (13, 14). β subunits of voltage-gated Ca²⁺ channels were repeatedly shown to be responsible for the facilitation of α₁ membrane insertion and to be potent modulators of α₁ current kinetics and voltage dependence (15, 16). Whether the loss of EC coupling in β₁-null mice was caused by decreased DHPR membrane expression or by the lack of a putative specific contribution of the β subunit to the skeletal muscle EC coupling apparatus (17, 18) was not clearly resolved. Recently, other β-functions were identified in skeletal muscle using the β₁-null mutant zebrafish relaxed (19, 20). Like the β₁-knock-out mouse (14) zebrafish relaxed is characterized by complete paralysis of skeletal muscle (21, 22). While β₁-knock-out mouse pups die immediately after birth due to respiratory paralysis (14), larvae of relaxed are able to survive for several days because of oxygen and metabolite diffusion via the skin (23). Using highly differentiated myotubes that are easy to isolate from these larvae, the lack of EC coupling could be described by quantitative immunocytochemistry as a moderate ∼50% reduction of α_1S membrane expression although α_1S charge movement was nearly absent, and, most strikingly, as the complete lack of the arrangement of DHPRs in tetrads (19). Thus, in skeletal muscle the β subunit enables EC coupling by (i) enhancing α_1S membrane targeting, (ii) facilitating α_1S charge movement, and (iii) enabling the ultrastructural arrangement of DHPRs in tetrads.The question arises, which of these functions are specific for the skeletal muscle β_1a and which ones are rather general properties of Ca²⁺ channel β subunits. Previous reconstitution studies made in the β₁-null mouse system (24, 25) using different β subunit constructs (26) did not allow differentiation between β-induced enhancement of non-functional α_1S membrane expression and the facilitation of α_1S charge movement, due to the lack of information on α_1S triad expression levels. Furthermore, the β-induced arrangement of DHPRs in tetrads was not detected as no ultrastructural information was obtained.In the present study, we established zebrafish mutant relaxed as an expression system to test different β subunits for their ability to restore skeletal muscle EC coupling. Using isolated myotubes for in vitro experiments (19, 27) and complete larvae for in vivo expression studies (28-31) and freeze-fracture electron microscopy, a clear differentiation between the major functional roles of β subunits was feasible in the zebrafish system. The cloned zebrafish β_1a and a mammalian (rabbit) β_1a were shown to completely restore all parameters of EC coupling when expressed in relaxed myotubes and larvae. However, the phylogenetically closest β subunit to β_1a, the cardiac/neuronal isoform β_2a from rat, as well as the ancestral β_M isoform from the housefly (Musca domestica), could recover functional α_1S membrane insertion, but led to very restricted tetrad formation when compared with β_1a, and thus to impaired DHPR-RyR1 coupling. This impairment caused drastic changes in skeletal muscle function.The present study shows that the enhancement of functional α_1S membrane expression is a common function of all the tested β subunits, from β_1a to even the most distant β_M, whereas the effective formation of tetrads and thus proper skeletal muscle EC coupling is an exclusive function of the skeletal muscle β_1a subunit. In context with previous studies, our results suggest a model according to which β_1a acts as an allosteric modifier of α_1S conformation. Only in the presence of β_1a, the α_1S subunit is properly folded to allow RyR1 anchoring and thus skeletal muscle-type EC coupling.     

Accommodating Discontinuities in Dimeric Left-Handed Coiled Coils in ATP Synthase External Stalks

ATP synthases from coupling membranes are complex rotary motors that convert the energy of proton gradients across coupling membranes into the chemical potential of the β-γ anhydride bond of ATP. Proton movement within the ring of c subunits localized in the F₀-sector drives γ and ɛ rotation within the F₁α₃β₃ catalytic core where substrates are bound and products are released. An external stalk composed of homodimeric subunits b₂ in Escherichia coli or heterodimeric bb′ in photosynthetic synthases connects F₀ subunit a with F₁ subunits δ and most likely α. The external stalk resists rotation, and is of interest both functionally and structurally. Hypotheses that the external stalk contributes to the overall efficiency of the reaction through elastic coupling of rotational substeps, and that stalks form staggered, right-handed coiled coils, are investigated here. We report on different structures that accommodate heptad discontinuities with either local or global underwinding. Analyses of the knob-and-hole packing of the E. coli b₂ and Synechocystis bb′ stalks strongly support the possibility that these proteins can adopt conventional left-handed coiled coils.     

1H NMR studies of the Fe7S8 ferredoxin from Bacillus schlegelii: a further attempt to understand Fe3S4 clusters

 The oxidized Fe₇S₈ ferredoxin from Bacillus schlegelii, containing both [Fe₃S₄]⁺ and [Fe₄S₄]²⁺ clusters, has been investigated by ¹H NMR spectroscopy. An extensive sequence-specific assignment of the hyperfine-shifted resonances has been obtained by making use of a computer-generated structural model. The pattern and the temperature dependence of the hyperfine shifts of the β-CH₂ protons of the cysteines coordinating the [Fe₃S₄]⁺ cluster are rationalized in terms of magnetic interactions between the iron ions. The same approach holds for the hyperfine coupling with ⁵⁷Fe. It is shown that the magnetic interactions are more asymmetric in Fe₇S₈ ferredoxins than in Fe₃S₄ ferredoxins. The NMR non-observability of the β-CH₂ protons of coordinated cysteines in the one-electron-reduced form has been discussed. Received: 19 June 1996 / Accepted: 2 August 1996