Structural basis for variant-specific neuroligin-binding by α-neurexin

Neurexins (Nrxs) are presynaptic membrane proteins with a single membrane-spanning domain that mediate asymmetric trans-synaptic cell adhesion by binding to their postsynaptic receptor neuroligins. α-Nrx has a large extracellular region comprised of multiple copies of laminin, neurexin, sex-hormone-binding globulin (LNS) domains and epidermal growth factor (EGF) modules, while that of β-Nrx has but a single LNS domain. It has long been known that the larger α-Nrx and the shorter β-Nrx show distinct binding behaviors toward different isoforms/variants of neuroligins, although the underlying mechanism has yet to be elucidated. Here, we describe the crystal structure of a fragment corresponding to the C-terminal one-third of the Nrx1α ectodomain, consisting of LNS5-EGF3-LNS6. The 2.3 Å-resolution structure revealed the presence of a domain configuration that was rigidified by inter-domain contacts, as opposed to the more common flexible “beads-on-a-string” arrangement. Although the neuroligin-binding site on the LNS6 domain was completely exposed, the location of the α-Nrx specific LNS5-EGF3 segment proved incompatible with the loop segment inserted in the B+ neuroligin variant, which explains the variant-specific neuroligin recognition capability observed in α-Nrx. This, combined with a low-resolution molecular envelope obtained by a single particle reconstruction performed on negatively stained full-length Nrx1α sample, allowed us to derive a structural model of the α-Nrx ectodomain. This model will help us understand not only how the large α-Nrx ectodomain is accommodated in the synaptic cleft, but also how the trans-synaptic adhesion mediated by α- and β-Nrxs could differentially affect synaptic structure and function.     

Molecular basis for the differential sensitivity of rat and human α9α10 nAChRs to α-conotoxin RgIA

J. Neurochem. (2012) 122, 1137-1144. ABSTRACT: The α9α10 nicotinic acetylcholine receptor (nAChR) may be a potential target in pathophysiology of the auditory system, chronic pain, and breast and lung cancers. Alpha-conotoxins, from the predatory marine snail Conus, are potent nicotinic antagonists, some of which are selective for the α9α10 nAChR. Here, we report a two order of magnitude species difference in the potency of α-conotoxin RgIA for the rat versus human α9α10 nAChR. We investigated the molecular mechanism of this difference. Heterologous expression of the rat α9 with the human α10 subunit in Xenopus oocytes resulted in a receptor that was blocked by RgIA with potency similar to that of the rat α9α10 nAChR. Conversely, expression of the human α9 with that of the rat α10 subunit resulted in a receptor that was blocked by RgIA with potency approaching that of the human α9α10 receptor. Systematic substitution of residues found in the human α9 subunit into the homologous position in the rat α9 subunit revealed that a single point mutation, Thr56 to Ile56, primarily accounts for this species difference. Remarkably, although the α9 nAChR subunit has previously been reported to provide the principal (+) binding face for binding of RgIA, Thr56 is located in the (-) complementary binding face.     

Structural basis of the selectivity of the β2-adrenergic receptor for fluorinated catecholamines

The important and diverse biological functions of adrenergic receptors, a subclass of G protein-coupled receptors (GPCRs), have made the search for compounds that selectively stimulate or inhibit the activity of different adrenergic receptor subtypes an important area of medicinal chemistry. We previously synthesized 2-, 5-, and 6-fluoronorepinehprine (FNE) and 2-, 5-, and 6-fluoroepinephrine (FEPI) and found that 2FNE and 2FEPI were selective β-adrenergic agonists and that 6FNE and 6FEPI were selective α-adrenergic agonists, while 5FNE and 5FEPI were unselective. Agonist potencies correlated well with receptor binding affinities. Here, through a combination of molecular modeling and site-directed mutagenesis, we have identified N293 in the β₂-adrenergic receptor as a crucial residue for the selectivity of the receptor for catecholamines fluorinated at different positions.     

Structural basis for μ-opioid receptor binding and activation

Opioids that stimulate the μ-opioid receptor (MOR1) are the most frequently prescribed and effective analgesics. Here we present a structural model of MOR1. Molecular dynamics simulations show a ligand-dependent increase in the conformational flexibility of the third intracellular loop that couples with the G protein complex. These simulations likewise identified residues that form frequent contacts with ligands. We validated the binding residues using site-directed mutagenesis coupled with radioligand binding and functional assays. The model was used to blindly screen a library of ～1.2 million compounds. From the 34 compounds predicted to be strong binders, the top three candidates were examined using biochemical assays. One compound showed high efficacy and potency. Post hoc testing revealed this compound to be nalmefene, a potent clinically used antagonist, thus further validating the model. In summary, the MOR1 model provides a tool for elucidating the structural mechanism of ligand-initiated cell signaling and for screening novel analgesics.     

N- and C-terminal extensions of μ-conotoxins increase potency and selectivity for neuronal sodium channels

μ-Conotoxins are peptide blockers of voltage-gated sodium channels (sodium channels), inhibiting tetrodotoxin-sensitive neuronal (Na(v) 1.2) and skeletal (Na(v) 1.4) subtypes with highest affinity. Structure-activity relationship studies of μ-conotoxins SIIIA, TIIIA, and KIIIA have shown that it is mainly the C-terminal part of the three-loop peptide that is involved in binding to the sodium channel. In this study, we characterize the effect of N- and C-terminal extensions of μ-conotoxins SIIIA, SIIIB, and TIIIA on their potency and selectivity for neuronal versus muscle sodium channels. Interestingly, extending the N- or C-terminal of the peptide by introducing neutral, positive, and/or negatively charged residues, the selectivity of the native peptide can be altered from neuronal to skeletal and the other way around. The results from this study provide further insight into the binding profile of μ-conotoxins at voltage-gated sodium channels, revealing that binding interactions outside the cysteine-stablilized loops can contribute to μ-conotoxin affinity and sodium channel selectivity.     

Structural basis for calcium and phosphatidylserine regulation of phospholipase C δ1

Many membrane-associated enzymes, including those of the phospholipase C (PLC) superfamily, are regulated by specific interactions with lipids. Previously, we have shown that the C2 domain of PLC δ1 is required for phosphatidylserine (PS)-dependent enzyme activation and that activation requires the presence of Ca(2+). To identify the site of interaction and the role of Ca(2+) in the activation mechanism, we mutagenized three highly conserved Ca(2+) binding residues (Asp-653, Asp-706, and Asp-708) to Gly in the C2 domain of PLC δ1. The PS-dependent Ca(2+) binding affinities of the mutant enzymes D653G, D706G, and D708G were reduced by 1 order of magnitude, and the maximal level of Ca(2+) binding was reduced to half of that of the native enzyme. The level of Ca(2+)-dependent PS binding was also reduced in the mutant enzymes. Under basal conditions, the Ca(2+) dependence and the maximal level of hydrolysis of phosphatidylinositol 4,5-bisphosphate were not altered in the mutants. However, the Ca(2+)-dependent PS stimulation was severely defective. PS reduces the K(m) of the native enzyme almost 20-fold, but far less for the mutants. Replacing Asp-653, Asp-706, and Asp-708 simultaneously with glycine in the C2 domain of PLC δ1 leads to a complete and selective loss of the stimulation and binding by PS. These results show that D653, D706, and D708 are required for Ca(2+) binding in the C2 domain and demonstrate a mechanism by which C2 domains can mediate regulation of enzyme activity by specific lipid ligands.     

Structural basis and kinetics of force-induced conformational changes of an αA domain-containing integrin

Background

Integrin α_Lβ₂ (lymphocyte function-associated antigen, LFA-1) bears force upon binding to its ligand intercellular adhesion molecule 1 (ICAM-1) when a leukocyte adheres to vascular endothelium or an antigen presenting cell (APC) during immune responses. The ligand binding propensity of LFA-1 is related to its conformations, which can be regulated by force. Three conformations of the LFA-1 αA domain, determined by the position of its α₇-helix, have been suggested to correspond to three different affinity states for ligand binding.

Methodology/Principal Findings

The kinetics of the force-driven transitions between these conformations has not been defined and dynamically coupled to the force-dependent dissociation from ligand. Here we show, by steered molecular dynamics (SMD) simulations, that the αA domain was successively transitioned through three distinct conformations upon pulling the C-terminus of its α₇-helix. Based on these sequential transitions, we have constructed a mathematical model to describe the coupling between the αA domain conformational changes of LFA-1 and its dissociation from ICAM-1 under force. Using this model to analyze the published data on the force-induced dissociation of single LFA-1/ICAM-1 bonds, we estimated the force-dependent kinetic rates of interstate transition from the short-lived to intermediate-lived and from intermediate-lived to long-lived states. Interestingly, force increased these transition rates; hence activation of LFA-1 was accelerated by pulling it via an engaged ICAM-1.

Conclusions/Significance

Our study defines the structural basis for mechanical regulation of the kinetics of LFA-1 αA domain conformational changes and relates these simulation results to experimental data of force-induced dissociation of single LFA-1/ICAM-1 bonds by a new mathematical model, thus provided detailed structural and kinetic characterizations for force-stabilization of LFA-1/ICAM-1 interaction.     

Importance of position 8 in μ-conotoxin KIIIA for voltage-gated sodium channel selectivity

μ-Conotoxin KIIIA from Conus kinoshitai is a 16-residue peptide that acts as a potent pore blocker of several voltage-gated sodium channels (Na(v)). In order to obtain more selective blockers and to investigate the role of Trp at position 8, we substituted this residue with Arg, Gln and Glu. KIIIA and analogues were tested on a range of Na(v) expressed in Xenopus laevis oocytes. The rank order of potency for KIIIA was: rNa(v)1.4 ≥ rNa(v)1.2 > mNa(v)1.6 > rNa(v)1.3, with IC(50) values of 48 ± 6 nm, 61 ± 5 nm, 183 ± 31 nm and 3.6 ± 0.3 μm, respectively, whereas no effect was seen on hNa(v)1.5 and hNa(v)1.8 at a concentration of 10 μm. Replacement of Trp8 resulted in more selective blockers with a preference for neuronal sodium channels over the skeletal sodium channel. The activity on rNa(v)1.4 was reduced about 40-, 70- and 200-fold for [W8R]KIIIA, [W8Q]KIIIA and [W8E]KIIIA, respectively. All analogues showed a completely reversible block of rNa(v)1.2, as opposed to the partial reversibility of KIIIA. At saturating concentrations, complete block of rNa(v)1.2 was never achieved. The residual current was lower than 10%, except for [W8E]KIIIA. KIIIA had no effect on the voltage dependence of activation of rNa(v)1.2, whereas all analogues caused a depolarizing shift. Overall, this study shows that Trp8 is a key residue in the pharmacophore. Replacement of Trp8 enables more selective blockers to be obtained for neuronal sodium channels. Trp is a key determinant for the reversibility of block of rNa(v)1.2.     

Structural basis of importin-α-mediated nuclear transport for Ku70 and Ku80

Ku70 and Ku80 form a heterodimeric complex involved in multiple nuclear processes. This complex plays a key role in DNA repair due to its ability to bind DNA double-strand breaks and facilitate repair by the nonhomologous end-joining pathway. Ku70 and Ku80 have been proposed to contain bipartite and monopartite nuclear localization sequences (NLSs), respectively, that allow them to be translocated to the nucleus independently of each other via the classical importin-α (Impα)/importin-β-mediated nuclear import pathway. To determine the structural basis of the recognition of Ku70 and Ku80 proteins by Impα, we solved the crystal structures of the complexes of Impα with the peptides corresponding to the Ku70 and Ku80 NLSs. Our structural studies confirm the binding of the Ku80 NLS as a classical monopartite NLS but reveal an unexpected binding mode for Ku70 NLS with only one basic cluster bound to the receptor. Both Ku70 and Ku80 therefore contain monopartite NLSs, and sequences outside the basic cluster make favorable interactions with Impα, suggesting that this may be a general feature in monopartite NLSs. We show that the Ku70 NLS has a higher affinity for Impα than the Ku80 NLS, consistent with more extensive interactions in its N-terminal region. The prospect of nuclear import of Ku70 and Ku80 independently of each other provides a powerful regulatory mechanism for the function of the Ku70/Ku80 heterodimer and independent functions of the two proteins.     

Structural basis for RNA-genome recognition during bacteriophage Qβ replication

Upon infection of Escherichia coli by bacteriophage Qβ, the virus-encoded β-subunit recruits host translation elongation factors EF-Tu and EF-Ts and ribosomal protein S1 to form the Qβ replicase holoenzyme complex, which is responsible for amplifying the Qβ (+)-RNA genome. Here, we use X-ray crystallography, NMR spectroscopy, as well as sequence conservation, surface electrostatic potential and mutational analyses to decipher the roles of the β-subunit and the first two oligonucleotide-oligosaccharide-binding domains of S1 (OB_1–2) in the recognition of Qβ (+)-RNA by the Qβ replicase complex. We show how three basic residues of the β subunit form a patch located adjacent to the OB₂ domain, and use NMR spectroscopy to demonstrate for the first time that OB₂ is able to interact with RNA. Neutralization of the basic residues by mutagenesis results in a loss of both the phage infectivity in vivo and the ability of Qβ replicase to amplify the genomic RNA in vitro. In contrast, replication of smaller replicable RNAs is not affected. Taken together, our data suggest that the β-subunit and protein S1 cooperatively bind the (+)-stranded Qβ genome during replication initiation and provide a foundation for understanding template discrimination during replication initiation.     

Structural and functional mapping of α-fetoprotein

Alpha-fetoprotein (AFP) is a major mammalian oncofetal protein, which is also present in small quantities in adults. It is a member of the albuminoid gene superfamily, which consists of AFP, serum albumin, vitamin D binding protein, and alpha-albumin (afamin). Although physicochemical and immunological properties of AFP have been well-studied, its biological role in embryo- and carcinogenesis and in adult organisms as well as mechanisms underlying its functioning remain unclear. During the recent decades, the biological role of AFP has been evaluated by identification of its functionally important sites. Comparison of primary structure of AFP and some physiologically active proteins revealed similarity of some polypeptide regions. This has been used for prediction of AFP functions (i.e., its multifunctionality). Localization of functionally important sites followed by determination of their amino acid composition and type of biological activity has provided valuable information for structural-functional mapping of AFP. Some peptide fragments of AFP have been synthesized and tested for biological activity. This review summarizes data on structural-functional interrelationships. We also describe functionally important AFP sites found by various groups during the last decade of structural-functional mapping of AFP with experimentally confirmed and putative biologically active sites.     

Structural basis for translation termination by archaeal RF1 and GTP-bound EF1�� complex

When a stop codon appears at the ribosomal A site, the class I and II release factors (RFs) terminate translation. In eukaryotes and archaea, the class I and II RFs form a heterodimeric complex, and complete the overall translation termination process in a GTP-dependent manner. However, the structural mechanism of the translation termination by the class I and II RF complex remains unresolved. In archaea, archaeal elongation factor 1 alpha (aEF1α), a carrier GTPase for tRNA, acts as a class II RF by forming a heterodimeric complex with archaeal RF1 (aRF1). We report the crystal structure of the aRF1·aEF1α complex, the first active class I and II RF complex. This structure remarkably resembles the tRNA·EF–Tu complex, suggesting that aRF1 is efficiently delivered to the ribosomal A site, by mimicking tRNA. It provides insights into the mechanism that couples GTP hydrolysis by the class II RF to stop codon recognition and peptidyl-tRNA hydrolysis by the class I RF. We discuss the different mechanisms by which aEF1α recognizes aRF1 and aPelota, another aRF1-related protein and molecular evolution of the three functions of aEF1α.     

Structural basis for the sequence-dependent effects of platinum–DNA adducts

The differences in efficacy and molecular mechanisms of platinum based anti-cancer drugs cisplatin (CP) and oxaliplatin (OX) have been hypothesized to be in part due to the differential binding affinity of cellular and damage recognition proteins to CP and OX adducts formed on adjacent guanines in genomic DNA. HMGB1a in particular exhibits higher binding affinity to CP-GG adducts, and the extent of discrimination between CP- and OX-GG adducts is dependent on the bases flanking the adducts. However, the structural basis for this differential binding is not known. Here, we show that the conformational dynamics of CP- and OX-GG adducts are distinct and depend on the sequence context of the adduct. Molecular dynamics simulations of the Pt-GG adducts in the TGGA sequence context revealed that even though the major conformations of CP- and OX-GG adducts were similar, the minor conformations were distinct. Using the pattern of hydrogen bond formation between the Pt–ammines and the adjacent DNA bases, we identified the major and minor conformations sampled by Pt–DNA. We found that the minor conformations sampled exclusively by the CP-GG adduct exhibit structural properties that favor binding by HMGB1a, which may explain its higher binding affinity to CP-GG adducts, while these conformations are not sampled by OX-GG adducts because of the constraints imposed by its cyclohexane ring, which may explain the negligible binding affinity of HMGB1a for OX-GG adducts in the TGGA sequence context. Based on these results, we postulate that the constraints imposed by the cyclohexane ring of OX affect the DNA conformations explored by OX-GG adduct compared to those of CP-GG adduct, which may influence the binding affinities of HMG-domain proteins for Pt-GG adducts, and that these conformations are further influenced by the DNA sequence context of the Pt-GG adduct.     

Somatic mutations in PI3Kα: Structural basis for enzyme activation and drug design

The PI3K pathway is a communication hub coordinating critical cell functions including cell survival, cell growth, proliferation, motility and metabolism. Because PI3Kα harbors recurrent somatic mutations resulting in gains of function in human cancers, it has emerged as an important drug target for many types of solid tumors. Various PI3K isoforms are also being evaluated as potential therapeutic targets for inflammation, heart disease, and hematological malignancies. Structural biology is providing insights into the flexibility of the PI3Ks, and providing basis for understanding the effects of mutations, drug resistance and specificity.     

Structural basis for the affinity of four insolubilized lectins,with a specificity for α-D-mannose,towards various glycopeptides with the N-glycosylamine linkage and related oligosaccharides

Precisions are given on the fine specificity and on the usefulness of immobilized concanavalin A,Lens culinaris, Vicia faba andPisum sativum agglutinins for fractionation of glycopeptides with the N-glycosylamine linkage.While insolubilized concanavalin A represents a very useful tool for the fractionation of both N-acetyllactosaminic and oligomannosidic type glycopeptides or related oligo-saccharides, immobilizedLens culinaris, as well asVicia faba orPisum sativum agglutinins allow the subfractionation of some N-acetyllactosaminic glycopeptide populations on the basis of the presence of an α-L-fucose residue substituting in C-6 position the N-acetylglucosamine residue involved in the N-glycosylamine bond.     

Structural features for α-galactomannan binding to galectin-1

Galectins have a highly conserved carbohydrate-binding domain to which a variety of galactose-containing saccharides, both β- and α-galactosides, can interact with varying degrees of affinity. Recently, we demonstrated that the relatively large α(1 → 6)-D-galacto-β(1 → 4)-D-mannan (Davanat) binds galectin-1 (gal-1) primarily at an alternative carbohydrate-binding domain. Here, we used a series of α-galactomannans (GMs) that vary in their mannose-to-galactose ratios for insight into an optimal structural signature for GM binding to gal-1. Heteronuclear single-quantum coherence nuclear magnetic resonance spectroscopy with (15)N-labeled gal-1 and statistical modeling suggest that the optimal signature consists of α-D-galactopyranosyl doublets surrounded by regions of about four or more "naked" mannose residues. These relatively large and complex GMs all appear to interact with varying degrees at essentially the same binding surface on gal-1 that includes the Davanat alternative binding site and elements of the canonical β-galactoside-binding region. The use of two small, well-defined GMs [6(1)-α(1 → 6)-D-galactosyl-β-D-mannotriaose and 6(3),6(4)-di-α(1 → 6)-D-galactosyl-β-D-mannopentaose] helped characterize how GMs, in general, interact in part with the canonical site. Overall, our findings contribute to better understanding interactions of gal-1 with larger, complex polysaccharides and to the development of GM-based therapeutics for clinical use.     

Structural basis for specificity of TGFβ family receptor small molecule inhibitors

Transforming growth factor-β (TGFβ) receptor kinase inhibitors have a great therapeutic potential. SB431542 is one of the mainly used kinase inhibitors of the TGFβ/Activin pathway receptors, but needs improvement of its EC₅₀ (EC₅₀ = 1 μM) to be translated to clinical use. A key feature of SB431542 is that it specifically targets receptors from the TGFβ/Activin pathway but not the closely related receptors from the bone morphogenic proteins (BMP) pathway. To understand the mechanisms of this selectivity, we solved the crystal structure of the TGFβ type I receptor (TβRI) kinase domain in complex with SB431542. We mutated TβRI residues coordinating SB431542 to their counterparts in activin-receptor like kinase 2 (ALK2), a BMP receptor kinase, and tested the kinase activity of mutated TβRI. We discovered that a Ser280Thr mutation yielded a TβRI variant that was resistant to SB431542 inhibition. Furthermore, the corresponding Thr283Ser mutation in ALK2 yielded a BMP receptor sensitive to SB431542. This demonstrated that Ser280 is the key determinant of selectivity for SB431542. This work provides a framework for optimising the SB431542 scaffold to more potent and selective inhibitors of the TGFβ/Activin pathway.     

Determination of L-AP4-bound human mGlu8 receptor amino terminal domain structure and the molecular basis for L-AP4’s group III mGlu receptor functional potency and selectivity

L-2-Amino-4-phosphonobutyric acid (L-AP4) is a known potent and selective agonist for the Group III mGlu receptors. However, it does not show any selectivity among the individual group III mGlu subtypes. In order to understand the molecular basis for this group selectivity, we solved the first human mGlu8 amino terminal domain (ATD) crystal structures in complex with L-glu and L-AP4. In comparison with other published L-glu-bound mGlu ATD structures, we have observed L-glu binds in a significantly different manner in mGlu1. Furthermore, these new structures provided evidence that both the electronic and steric nature of the distal phosphate of L-AP4 contribute to its exquisite Group III functional agonist potency and selectivity.