Crystal Structures of the Human G3BP1 NTF2-Like Domain Visualize FxFG Nup Repeat Specificity

Ras GTPase Activating Protein SH3 Domain Binding Protein (G3BP) is a potential anti-cancer drug target implicated in several cellular functions. We have used protein crystallography to solve crystal structures of the human G3BP1 NTF2-like domain both alone and in complex with an FxFG Nup repeat peptide. Despite high structural similarity, the FxFG binding site is located between two alpha helices in the G3BP1 NTF2-like domain and not at the dimer interface as observed for nuclear transport factor 2. ITC studies showed specificity towards the FxFG motif but not FG and GLFG motifs. The unliganded form of the G3BP1 NTF2-like domain was solved in two crystal forms to resolutions of 1.6 and 3.3 Å in space groups P2₁2₁2₁ and P6₃22 based on two different constructs, residues 1–139 and 11–139, respectively. Crystal packing of the N-terminal residues against a symmetry related molecule in the P2₁2₁2₁ crystal form might indicate a novel ligand binding site that, however, remains to be validated. The crystal structures give insight into the nuclear transportation mechanisms of G3BP and provide a basis for future structure based drug design.     

Crystal structures of IFT70/52 and IFT52/46 provide insight into intraflagellar transport B core complex assembly

Cilia are microtubule-based organelles that assemble via intraflagellar transport (IFT) and function as signaling hubs on eukaryotic cells. IFT relies on molecular motors and IFT complexes that mediate the contacts with ciliary cargo. To elucidate the architecture of the IFT-B complex, we reconstituted and purified the nonameric IFT-B core from Chlamydomonas reinhardtii and determined the crystal structures of C. reinhardtii IFT70/52 and Tetrahymena IFT52/46 subcomplexes. The 2.5-Å resolution IFT70/52 structure shows that IFT52_330–370 is buried deeply within the IFT70 tetratricopeptide repeat superhelix. Furthermore, the polycystic kidney disease protein IFT88 binds IFT52_281–329 in a complex that interacts directly with IFT70/IFT52_330–381 in trans. The structure of IFT52C/IFT46C was solved at 2.3 Å resolution, and we show that it is essential for IFT-B core integrity by mediating interaction between IFT88/70/52/46 and IFT81/74/27/25/22 subcomplexes. Consistent with this, overexpression of mammalian IFT52C in MDCK cells is dominant-negative and causes IFT protein mislocalization and disrupted ciliogenesis. These data further rationalize several ciliogenesis phenotypes of IFT mutant strains.     

Structural Determination of Functional Units of the Nucleotide Binding Domain (NBD94) of the Reticulocyte Binding Protein Py235 of Plasmodium yoelii

Background

Invasion of the red blood cells (RBC) by the merozoite of malaria parasites involves a large number of receptor ligand interactions. The reticulocyte binding protein homologue family (RH) plays an important role in erythrocyte recognition as well as virulence. Recently, it has been shown that members of RH in addition to receptor binding may also have a role as ATP/ADP sensor. A 94 kDa region named Nucleotide-Binding Domain 94 (NBD94) of Plasmodium yoelii YM, representative of the putative nucleotide binding region of RH, has been demonstrated to bind ATP and ADP selectively. Binding of ATP or ADP induced nucleotide-dependent structural changes in the C-terminal hinge-region of NBD94, and directly impacted on the RBC binding ability of RH.

Methodology/Principal Findings

In order to find the smallest structural unit, able to bind nucleotides, and its coupling module, the hinge region, three truncated domains of NBD94 have been generated, termed NBD94_444–547, NBD94_566–663 and NBD94_674–793, respectively. Using fluorescence correlation spectroscopy NBD94_444–547 has been identified to form the smallest nucleotide binding segment, sensitive for ATP and ADP, which became inhibited by 4-Chloro-7-nitrobenzofurazan. The shape of NBD94_444–547 in solution was calculated from small-angle X-ray scattering data, revealing an elongated molecule, comprised of two globular domains, connected by a spiral segment of about 73.1 Å in length. The high quality of the constructs, forming the hinge-region, NBD94_566–663 and NBD94_674–793 enabled to determine the first crystallographic and solution structure, respectively. The crystal structure of NBD94_566–663 consists of two helices with 97.8 Å and 48.6 Å in length, linked by a loop. By comparison, the low resolution structure of NBD94_674–793 in solution represents a chair–like shape with three architectural segments.

Conclusions

These structures give the first insight into how nucleotide binding impacts on the overall structure of RH and demonstrates the potential use of this region as a novel drug target.     

B-form to A-form conversion by a 3'-terminal ribose: crystal structure of the chimera d(CCACTAGTG)r(G)

The crystal structure of the chimerical decamer d(CCACTAGTG)r(G), bearing a 3′-terminal ribo-guanidine, has been solved and refined at 1.8 Å resolution (R-factor 16.6%; free R-factor 22.8%). The decamer crystallizes in the orthorhombic space group P2₁2₁2₁ with unit cell constants a = 23.90 Å, b = 45.76 Å and c = 49.27 Å. The structure was solved by molecular replacement using the coordinates of the isomorphous chimera r(GCG)d(TATACGC). The final model contains one duplex and 77 water molecules per asymmetric unit. Surprisingly, all residues adopt a conformation typical for A-form nucleic acids (C3′-endo type sugar pucker) although the all-DNA analog, d(CCACTAGTGG), has been crystallized in the B-form. Comparing circular dichroism spectra of the chimera and the corresponding all-DNA sequence reveals a similar trend of the former molecule to adopt an A-like conformation in solution. The results suggest that the preference of ribonucleotides for the A-form is communicated into the 5′-direction of an oligonucleotide strand, although direct interactions of the 2′-hydroxyl group can only be discerned with nucleotides in the 3′-direction of a C3′-endo puckered ribose. These observations imply that forces like water-mediated contacts, the concerted motions of backbone torsion angles, and stacking preferences, are responsible for such long-range influences. This bi-directional structural communication originating from a ribonucleotide can be expected to contribute to the stability of the A-form within all-RNA duplexes.     

Synthesis and crystal structure of an octamer RNA r(guguuuac)/r(guaggcac) with G.G/U.U tandem wobble base pairs: comparison with other tandem G.U pairs

We have determined the crystal structure of the RNA octamer duplex r(guguuuac)/r(guaggcac) with a tandem wobble pair, G·G/U·U (motif III), to compare it with U·G/G·U (motif I) and G·U/U·G (motif II) and to better understand their relative stabilities. The crystal belongs to the rhombohedral space group R3. The hexagonal unit cell dimensions are a = b = 41.92 Å, c = 56.41 Å, and γ = 120°, with one duplex in the asymmetric unit. The structure was solved by the molecular replacement method at 1.9 Å resolution and refined to a final R factor of 19.9% and R_free of 23.3% for 2862 reflections in the resolution range 10.0–1.9 Å with F ≥ 2σ(F). The final model contains 335 atoms for the RNA duplex and 30 water molecules. The A-RNA stacks in the familiar head-to-tail fashion forming a pseudo-continuous helix. The uridine bases of the tandem U·G pairs have slipped towards the minor groove relative to the guanine bases and the uridine O2 atoms form bifurcated hydrogen bonds with the N1 and N2 of guanines. The N2 of guanine and O2 of uridine do not bridge the ‘locked’ water molecule in the minor groove, as in motifs I and II, but are bridged by water molecules in the major groove. A comparison of base stacking stabilities of motif III with motifs I and II confirms the result of thermodynamic studies, motif I > motif III > motif II.     

Flexibility between the Protease and Helicase Domains of the Dengue Virus NS3 Protein Conferred by the Linker Region and Its Functional Implications

The dengue virus (DENV) NS3 protein is essential for viral polyprotein processing and RNA replication. It contains an N-terminal serine protease region (residues 1–168) joined to an RNA helicase (residues 180–618) by an 11-amino acid linker (169–179). The structure at 3.15 Å of the soluble NS3 protein from DENV4 covalently attached to 18 residues of the NS2B cofactor region (NS2B₁₈NS3) revealed an elongated molecule with the protease domain abutting subdomains I and II of the helicase (Luo, D., Xu, T., Hunke, C., Grüber, G., Vasudevan, S. G., and Lescar, J. (2008) J. Virol. 82, 173–183). Unexpectedly, using similar crystal growth conditions, we observed an alternative conformation where the protease domain has rotated by ∼161° with respect to the helicase domain. We report this new crystal structure bound to ADP-Mn²⁺ refined to a resolution of 2.2 Å. The biological significance for interdomain flexibility conferred by the linker region was probed by either inserting a Gly residue between Glu¹⁷³ and Pro¹⁷⁴ or replacing Pro¹⁷⁴ with a Gly residue. Both mutations resulted in significantly lower ATPase and helicase activities. We next increased flexibility in the linker by introducing a Pro¹⁷⁶ to Gly mutation in a DENV2 replicon system. A 70% reduction in luciferase reporter signal and a similar reduction in the level of viral RNA synthesis were observed. Our results indicate that the linker region has evolved to an optimum length to confer flexibility to the NS3 protein that is required both for polyprotein processing and RNA replication.     

Mutations in PBP2 from ceftriaxone-resistant Neisseria gonorrhoeae alter the dynamics of the β3–β4 loop to favor a low-affinity drug-binding state

Resistance to the extended-spectrum cephalosporin ceftriaxone in the pathogenic bacteria Neisseria gonorrhoeae is conferred by mutations in penicillin-binding protein 2 (PBP2), the lethal target of the antibiotic, but how these mutations exert their effect at the molecular level is unclear. Using solution NMR, X-ray crystallography, and isothermal titration calorimetry, we report that WT PBP2 exchanges dynamically between a low-affinity state with an extended β3–β4 loop conformation and a high-affinity state with an inward β3–β4 loop conformation. Histidine-514, which is located at the boundary of the β4 strand, plays an important role during the exchange between these two conformational states. We also find that mutations present in PBP2 from H041, a ceftriaxone-resistant strain of N. gonorrhoeae, increase resistance to ceftriaxone by destabilizing the inward β3–β4 loop conformation or stabilizing the extended β3–β4 loop conformation to favor the low-affinity drug-binding state. These observations reveal a unique mechanism for ceftriaxone resistance, whereby mutations in PBP2 lower the proportion of target molecules in the high-affinity drug-binding state and thus reduce inhibition at lower drug concentrations.Keywords: PBP2, Neisseria gonorrhoeae, beta-lactam, conformational dynamics, antibiotic resistance

Neisseria gonorrhoeae is the causative agent of the sexually transmitted infection gonorrhea, with nearly 80 million cases worldwide each year (1). Without antibiotic treatment, infections persist as a chronic disease and can cause serious sequelae, including pelvic inflammatory disease, infertility, arthritis, and disseminated infections (2). For many years, N. gonorrhoeae was treated with a single dose of penicillin, and more recently, ceftriaxone. In 2012, the emergence of several high-level ceftriaxone-resistant strains led the Centers for Disease Control and Prevention to change its recommended treatment for gonorrhea from monotherapy to dual therapy with ceftriaxone and azithromycin (3, 4, 5). However, treatment failures have been reported for both agents, and in 2018, a strain with high-level resistance to both ceftriaxone and azithromycin was identified (6, 7). Concern about azithromycin resistance led the Centers for Disease Control and Prevention recently to drop the recommendation of dual therapy in favor of an increased dose (500 mg) of ceftriaxone alone (8). Both penicillin and ceftriaxone inhibit cell wall biosynthesis in N. gonorrhoeae by targeting penicillin-binding protein 2 (PBP2).PBP2 is an essential peptidoglycan transpeptidase (TPase) that crosslinks the peptide chains from adjacent peptidoglycan strands during cell-wall synthesis (9). β-lactam antibiotics, including the extended-spectrum cephalosporin (ESC) ceftriaxone, are analogs of the d-Ala-d-Ala C terminus of the peptidoglycan substrate and as such target PBP2 by binding to and reacting with the active-site serine nucleophile (Ser310 in N. gonorrhoeae PBP2) to form a covalently acylated complex (10, 11). The acylation reaction (Equation 1) proceeds first through formation of a noncovalent complex with the β-lactam (defined by the equilibrium constant, Ks), which is then attacked by the serine nucleophile to form a covalent acyl-enzyme complex (k₂). For PBPs, hydrolysis of the acyl-enzyme (k₃) is very slow compared with its formation, and the enzyme is essentially irreversibly inactivated. The acylation of PBPs by β-lactam antibiotics is therefore defined by a second-order rate constant, k₂/Ks (M⁻¹ s⁻¹), which reflects both the noncovalent binding affinity (Ks) and the first-order acylation rate (k₂):E+S↔KsE⋅S→k2E−S→k3E+P(1)The emergence of resistance to penicillin and ceftriaxone in N. gonorrhoeae occurs primarily via the acquisition of mutant alleles of the penA gene encoding PBP2 (12). These alleles are referred to as mosaic because they arise through multiple homologous recombination events with DNA released by commensal Neisseria species. PBP2 from the high-level ceftriaxone-resistant strain, H041, contains 61 mutations compared with PBP2 from the antibiotic-susceptible strain, FA19 (13, 14). Determining how these mutations lower the k₂/Ks of ceftriaxone for PBP2 by over 10,000-fold while still preserving essential TPase activity is fundamental for understanding the evolution of antibiotic resistance.Toward this goal, we have identified a subset of these mutations that, when incorporated into the penA gene from FA19, confer ∼80% of the increase in minimum inhibitory concentration for ceftriaxone relative to that of the penA gene from H041 (penA41) (15, 16). We recently reported the structures of apo and ceftriaxone-acylated PBP2 at high resolution and have detailed conformational changes in β3 and the β3–β4 loop involved in antibiotic binding and acylation (17). Intriguingly, although present in the active site region, most of the mutations conferring resistance are not in direct contact with ceftriaxone in the crystal structure of acylated PBP2 (17, 18). We have proposed that these mutations alter the binding and acylation kinetics of PBP2 with ceftriaxone by restricting protein dynamics (18).To understand further the structural and biochemical mechanisms by which these mutations lower the acylation rates of β-lactam antibiotics, we utilized a combination of solution ¹⁹F NMR, X-ray crystallography, and biochemical approaches to investigate PBP2. We report that the β3–β4 loop in the TPase domain of WT PBP2, which is known to adopt markedly different conformations in the apo versus acylated crystal structures (17), samples two major conformational states in solution. Substitutions of WT PBP2 residues with mutations in H041 that confer ceftriaxone resistance alter the conformational landscape of PBP2 by destabilizing the high-affinity state containing the inward conformation of the β3–β4 loop and stabilizing a low-affinity conformation containing an extended β3–β4 loop conformation, thereby restricting access to the inward conformation required for high-affinity drug binding. Our combined solution NMR and crystallographic analyses of PBP2 and its preacylation drug complexes further support the notion that mutations in PBP2 from ceftriaxone-resistant strains of N. gonorrhoeae confer antibiotic resistance by hindering conformational changes required to form a productive drug-binding state (18).     

Structure of the Sec13–Sec16 edge element,a template for assembly of the COPII vesicle coat

Ancestral coatomer element 1 (ACE1) proteins assemble latticework coats for COPII vesicles and the nuclear pore complex. The ACE1 protein Sec31 and Sec13 make a 2:2 tetramer that forms the edge element of the COPII outer coat. In this study, we report that the COPII accessory protein Sec16 also contains an ACE1. The 165-kD crystal structure of the central domain of Sec16 in complex with Sec13 was solved at 2.7-Å resolution. Sec16 and Sec13 also make a 2:2 tetramer, another edge element for the COPII system. Domain swapping at the ACE1–ACE1 interface is observed both in the prior structure of Sec13–Sec31 and in Sec13–Sec16. A Sec31 mutant in which domain swapping is prevented adopts an unprecedented laminated structure, solved at 2.8-Å resolution. Our in vivo data suggest that the ACE1 element of Sec31 can functionally replace the ACE1 element of Sec16. Our data support Sec16 as a scaffold for the COPII system and a template for the Sec13–Sec31 coat.     

C3larvin Toxin,an ADP-ribosyltransferase from Paenibacillus larvae

C3larvin toxin was identified by a bioinformatic strategy as a putative mono-ADP-ribosyltransferase and a possible virulence factor from Paenibacillus larvae, which is the causative agent of American Foulbrood in honey bees. C3larvin targets RhoA as a substrate for its transferase reaction, and kinetics for both the NAD⁺ (K_m = 34 ± 12 μm) and RhoA (K_m = 17 ± 3 μm) substrates were characterized for this enzyme from the mono-ADP-ribosyltransferase C3 toxin subgroup. C3larvin is toxic to yeast when expressed in the cytoplasm, and catalytic variants of the enzyme lost the ability to kill the yeast host, indicating that the toxin exerts its lethality through its enzyme activity. A small molecule inhibitor of C3larvin enzymatic activity was discovered called M3 (K_i = 11 ± 2 μm), and to our knowledge, is the first inhibitor of transferase activity of the C3 toxin family. C3larvin was crystallized, and its crystal structure (apoenzyme) was solved to 2.3 Å resolution. C3larvin was also shown to have a different mechanism of cell entry from other C3 toxins.     

Synthesis,Crystal Structure and Thermal Decomposition of the New Cadmium Selenite Chloride,Cd4(SeO3)2OCl2

A synthetic study in the Cd-Se-O-Cl system led to formation of the new oxochloride compound Cd₄(SeO₃)₂OCl₂ via solid state reactions. The compound crystallizes in the orthorhombic space group Fmmm with cell parameters a = 7.3610(3) Å, b = 15.4936(2) Å, c = 17.5603(3) Å, Z = 8, S = 0.969, F(000) = 2800, R = 0.0185, R_w = 0.0384. Single crystal X-ray data were collected at 293 K. The crystal structure can be considered as layered and the building units are distorted [Cd(1)O₆] octahedra, distorted [Cd(2)O₈] cubes, irregular [Cd(3)O₄Cl₂] polyhedra and SeO₃E trigonal pyramids. There are two crystallographically unique Cl atoms that both are half occupied. Thermogravimetric studies show that the compound starts to decompose at 500°C. The crystal structure of the new compound is closely related to the previously described compound Cd₄(SeO₃)₂Cl₄(H₂O).     

The Symmetrical Structure of Structural Maintenance of Chromosomes (SMC) and MukB Proteins: Long,Antiparallel Coiled Coils,Folded at a Flexible Hinge

Structural maintenance of chromosomes (SMC) proteins function in chromosome condensation and several other aspects of DNA processing. They are large proteins characterized by an NH₂-terminal nucleotide triphosphate (NTP)-binding domain, two long segments of coiled coil separated by a hinge, and a COOH-terminal domain. Here, we have visualized by EM the SMC protein from Bacillus subtilis (BsSMC) and MukB from Escherichia coli, which we argue is a divergent SMC protein. Both BsSMC and MukB show two thin rods with globular domains at the ends emerging from the hinge. The hinge appears to be quite flexible: the arms can open up to 180°, separating the terminal domains by 100 nm, or close to near 0°, bringing the terminal globular domains together.A surprising observation is that the ∼300–amino acid–long coiled coils are in an antiparallel arrangement. Known coiled coils are almost all parallel, and the longest antiparallel coiled coils known previously are 35–45 amino acids long. This antiparallel arrangement produces a symmetrical molecule with both an NH₂- and a COOH-terminal domain at each end. The SMC molecule therefore has two complete and identical functional domains at the ends of the long arms. The bifunctional symmetry and a possible scissoring action at the hinge should provide unique biomechanical properties to the SMC proteins.     

The ACTN3 R577X Polymorphism Is Associated with Cardiometabolic Fitness in Healthy Young Adults

Homozygosity for a premature stop codon (X) in the ACTN3 “sprinter” gene is common in humans despite the fact that it reduces muscle size, strength and power. Because of the close relationship between skeletal muscle function and cardiometabolic health we examined the influence of ACTN3 R577X polymorphism over cardiovascular and metabolic characteristics of young adults (n = 98 males, n = 102 females; 23 ± 4.2 years) from our Assessing Inherent Markers for Metabolic syndrome in the Young (AIMMY) study. Both males and females with the RR vs XX genotype achieved higher mean VO₂ peak scores (47.8 ± 1.5 vs 43.2 ±1.8 ml/O₂/min, p = 0.002) and exhibited higher resting systolic (115 ± 2 vs 105 ± mmHg, p = 0.027) and diastolic (69 ± 3 vs 59 ± 3 mmHg, p = 0.005) blood pressure suggesting a role for ACTN3 in the maintenance of vascular tone. We subsequently identified the expression of alpha-actinin 3 protein in pulmonary artery smooth muscle, which may explain the genotype-specific differences in cardiovascular adaptation to acute exercise. In addition, we utilized targeted serum metabolomics to distinguish between RR and XX genotypes, suggesting an additional role for the ACTN3 R577X polymorphism in human metabolism. Taken together, these results identify significant cardiometabolic effects associated with possessing one or more functional copies of the ACTN3 gene.     

Solution structure of psi32-modified anticodon stem-loop of Escherichia coli tRNAPhe

Nucleoside base modifications can alter the structures and dynamics of RNA molecules and are important in tRNAs for maintaining translational fidelity and efficiency. The unmodified anticodon stem–loop from Escherichia coli tRNA^Phe forms a trinucleotide loop in solution, but Mg²⁺ and dimethylallyl modification of A₃₇ N6 destabilize the loop-proximal base pairs and increase the mobility of the loop nucleotides. The anticodon arm has three additional modifications, ψ₃₂, ψ₃₉, and A₃₇ C2-thiomethyl. We have used NMR spectroscopy to investigate the structural and dynamical effects of ψ₃₂ on the anticodon stem-loop from E.coli tRNA^Phe. The ψ₃₂ modification does not significantly alter the structure of the anticodon stem–loop relative to the unmodified parent molecule. The stem of the RNA molecule includes base pairs ψ₃₂-A₃₈ and U₃₃–A₃₇ and the base of ψ₃₂ stacks between U₃₃ and A₃₁. The glycosidic bond of ψ₃₂ is in the anti configuration and is paired with A₃₈ in a Watson–Crick geometry, unlike residue 32 in most crystal structures of tRNA. The ψ₃₂ modification increases the melting temperature of the stem by ~3.5°C, although the ψ₃₂ and U₃₃ imino resonances are exchange broadened. The results suggest that ψ₃₂ functions to preserve the stem integrity in the presence of additional loop modifications or after reorganization of the loop into a translationally functional conformation.     

Fission yeast Duf89 and Duf8901 are cobalt/nickel-dependent phosphatase–pyrophosphatases that act via a covalent aspartyl–phosphate intermediate

Domain of Unknown Function 89 (DUF89) proteins are metal-dependent phosphohydrolases. Exemplary DUF89 enzymes differ in their metal and phosphosubstrate preferences. Here, we interrogated the activities and structures of two DUF89 paralogs from fission yeast—Duf89 and Duf8901. We find that Duf89 and Duf8901 are cobalt/nickel-dependent phosphohydrolases adept at hydrolyzing p-nitrophenylphosphate and PP_i. Crystal structures of metal-free Duf89 and Co²⁺-bound Duf8901 disclosed two enzyme conformations that differed with respect to the position of a three-helix module, which is either oriented away from the active site in Duf89 or forms a lid over the active site in Duf8901. Lid closure results in a 16 Å movement of Duf8901 Asp195, vis-à-vis Asp199 in Duf89, that brings Asp195 into contact with an octahedrally coordinated cobalt. Reaction of Duf8901 with BeCl₂ and NaF in the presence of divalent cations Co²⁺, Ni²⁺, or Zn²⁺ generated covalent Duf8901-(Asp248)–beryllium trifluoride (BeF₃)•Co²⁺, Duf8901-(Asp248)–BeF₃•Ni²⁺, or Duf8901-(Asp248)–BeF₃•Zn²⁺ adducts, the structures of which suggest a two-step catalytic mechanism via formation and hydrolysis of an enzyme-(aspartyl)–phosphate intermediate. Alanine mutations of Duf8901 Asp248, Asn249, Lys401, Asp286, and Asp195 that interact with BeF₃•Co²⁺ squelched p-nitrophenylphosphatase activity. A 1.8 Å structure of a Duf8901-(Asp248)–AlF₄–OH₂•Co²⁺ transition-state mimetic suggests an associative mechanism in which Asp195 and Asp363 orient and activate the water nucleophile. Whereas deletion of the duf89 gene elicited a phenotype in which expression of phosphate homeostasis gene pho1 was derepressed, deleting duf8901 did not, thereby hinting that the DUF89 paralogs have distinct functional repertoires in vivo.     

Structural insights into the function-modulating effects of nanobody binding to the integrin receptor αMβ2

The integrin receptor α_Mβ₂ mediates phagocytosis of complement-opsonized objects, adhesion to the extracellular matrix, and transendothelial migration of leukocytes. However, the mechanistic aspects of α_Mβ₂ signaling upon ligand binding are unclear. Here, we present the first atomic structure of the human α_Mβ₂ headpiece fragment in complex with the nanobody (Nb) hCD11bNb1 at a resolution of 3.2 Å. We show that the receptor headpiece adopts the closed conformation expected to exhibit low ligand affinity. The crystal structure indicates that in the R77H α_M variant, associated with systemic lupus erythematosus, the modified allosteric relationship between ligand binding and integrin outside–inside signaling is due to subtle conformational effects transmitted over a distance of 40 Å. Furthermore, we found the Nb binds to the αI domain of the α_M subunit in an Mg²⁺-independent manner with low nanomolar affinity. Biochemical and biophysical experiments with purified proteins demonstrated that the Nb acts as a competitive inhibitor through steric hindrance exerted on the thioester domain of complement component iC3b attempting to bind the α_M subunit. Surprisingly, we show that the Nb stimulates the interaction of cell-bound α_Mβ₂ with iC3b, suggesting that it may represent a novel high-affinity proteinaceous α_Mβ₂-specific agonist. Taken together, our data suggest that the iC3b–α_Mβ₂ complex may be more dynamic than predicted from the crystal structure of the core complex. We propose a model based on the conformational spectrum of the receptor to reconcile these observations regarding the functional consequences of hCD11bNb1 binding to α_Mβ₂.     

Structural insights into a flavin-dependent dehalogenase HadA explain catalysis and substrate inhibition via quadruple π-stacking

HadA is a flavin-dependent monooxygenase catalyzing hydroxylation plus dehalogenation/denitration, which is useful for biodetoxification and biodetection. In this study, the X-ray structure of wild-type HadA (HadA_WT) co-complexed with reduced FAD (FADH^–) and 4-nitrophenol (4NP) (HadA_WT−FADH^–−4NP) was solved at 2.3-Å resolution, providing the first full package (with flavin and substrate bound) structure of a monooxygenase of this type. Residues Arg101, Gln158, Arg161, Thr193, Asp254, Arg233, and Arg439 constitute a flavin-binding pocket, whereas the 4NP-binding pocket contains the aromatic side chain of Phe206, which provides π-π stacking and also is a part of the hydrophobic pocket formed by Phe155, Phe286, Thr449, and Leu457. Based on site-directed mutagenesis and stopped-flow experiments, Thr193, Asp254, and His290 are important for C4a-hydroperoxyflavin formation with His290, also serving as a catalytic base for hydroxylation. We also identified a novel structural motif of quadruple π-stacking (π-π-π-π) provided by two 4NP and two Phe441 from two subunits. This motif promotes 4NP binding in a nonproductive dead-end complex, which prevents C4a-hydroperoxy-FAD formation when HadA is premixed with aromatic substrates. We also solved the structure of the HadA_Phe441Val−FADH^–−4NP complex at 2.3-Å resolution. Although 4NP can still bind to this variant, the quadruple π-stacking motif was disrupted. All HadA_Phe441 variants lack substrate inhibition behavior, confirming that quadruple π-stacking is a main cause of dead-end complex formation. Moreover, the activities of these HadA_Phe441 variants were improved by ⁓20%, suggesting that insights gained from the flavin-dependent monooxygenases illustrated here should be useful for future improvement of HadA’s biocatalytic applications.     

Cardio-Respiratory Reference Data in 4631 Healthy Men and Women 20-90 Years: The HUNT 3 Fitness Study

Purpose

To provide a large reference material on key cardio-respiratory variables in a healthy population of Norwegian men and women aged 20–90 years.

Methods

Sub maximal and peak levels of cardio-respiratory variables were measured using cardiopulmonary exercise testing during treadmill running.

Results

The highest peak ventilation among men (141.9±24.5 L·min⁻¹) and women (92.0±16.5 L·min⁻¹) was observed in the youngest age group (20–29 years, sex differences p<0.001) with an average 7% reduction per decade. The highest tidal volumes were observed in the 30–39 and 40–49 year age groups among men (2.94±0.46 L) and women (2.06±0.32 L) (sex differences p<0.001), with a subsequent average 6% reduction per decade. Ventilatory threshold and respiratory compensation point were observed at approximately 77% and 87% of peak oxygen uptake (VO_2peak) among men and women, respectively. The best ventilatory efficiency (EqVCO_2Than) was observed in the youngest age group (20–29 years) in both men (26.2±2.8) and woman (27.5±2.7) (sex differences p<0.001) with an average 3% deterioration in ventilatory efficiency per decade.

Conclusion

This is the largest European reference material of cardio-respiratory variables in healthy men and women aged 20–90 years, establishing normal values for, and associations between key cardio-respiratory parameters. This will be useful in clinical decision making when evaluating cardiopulmonary health in similar populations.     

Structural Basis for a Munc13–1 Homodimer to Munc13–1/RIM Heterodimer Switch

C ₂ domains are well characterized as Ca ²⁺/phospholipid-binding modules, but little is known about how they mediate protein–protein interactions. In neurons, a Munc13–1 C ₂A-domain/RIM zinc-finger domain (ZF) heterodimer couples synaptic vesicle priming to presynaptic plasticity. We now show that the Munc13–1 C ₂A domain homodimerizes, and that homodimerization competes with Munc13–1/RIM heterodimerization. X-ray diffraction studies guided by nuclear magnetic resonance (NMR) experiments reveal the crystal structures of the Munc13–1 C ₂A-domain homodimer and the Munc13–1 C ₂A-domain/RIM ZF heterodimer at 1.44 Å and 1.78 Å resolution, respectively. The C ₂A domain adopts a β-sandwich structure with a four-stranded concave side that mediates homodimerization, leading to the formation of an eight-stranded β-barrel. In contrast, heterodimerization involves the bottom tip of the C ₂A-domain β-sandwich and a C-terminal α-helical extension, which wrap around the RIM ZF domain. Our results describe the structural basis for a Munc13–1 homodimer–Munc13–1/RIM heterodimer switch that may be crucial for vesicle priming and presynaptic plasticity, uncovering at the same time an unexpected versatility of C ₂ domains as protein–protein interaction modules, and illustrating the power of combining NMR spectroscopy and X-ray crystallography to study protein complexes.     

Development of 3-methyl/3-(morpholinomethyl)benzofuran derivatives as novel antitumor agents towards non-small cell lung cancer cells

As one of the most lethal malignancies, lung cancer is considered to account for approximately one-fifth of all malignant tumours-related deaths worldwide. This study reports the synthesis and in vitro biological assessment of two sets of 3-methylbenzofurans (4a–d, 6a–c, 8a–c and 11) and 3-(morpholinomethyl)benzofurans (15a–c, 16a–b, 17a–b and 18) as potential anticancer agents towards non-small cell lung carcinoma A549 and NCI-H23 cell lines, with VEGFR-2 inhibitory activity. The target benzofuran-based derivatives efficiently inhibited the growth of both A549 and NCI-H23 cell lines with IC₅₀ spanning in ranges 1.48–47.02 and 0.49–68.9 µM, respectively. The three most active benzofurans (4b, 15a and 16a) were further investigated for their effects on the cell cycle progression and apoptosis in A549 (for 4b) and NCI-H23 (for 15a and 16a) cell lines. Furthermore, benzofurans 4b, 15a and 16a displayed good VEGFR-2 inhibitory activity with IC₅₀ equal 77.97, 132.5 and 45.4 nM, respectively.