Insight into the Structure of the “Unstructured” Tau Protein

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Crystal Structure and Molecular Imaging of the Nav Channel β3 Subunit Indicates a Trimeric Assembly

The vertebrate sodium (Na_v) channel is composed of an ion-conducting α subunit and associated β subunits. Here, we report the crystal structure of the human β3 subunit immunoglobulin (Ig) domain, a functionally important component of Na_v channels in neurons and cardiomyocytes. Surprisingly, we found that the β3 subunit Ig domain assembles as a trimer in the crystal asymmetric unit. Analytical ultracentrifugation confirmed the presence of Ig domain monomers, dimers, and trimers in free solution, and atomic force microscopy imaging also detected full-length β3 subunit monomers, dimers, and trimers. Mutation of a cysteine residue critical for maintaining the trimer interface destabilized both dimers and trimers. Using fluorescence photoactivated localization microscopy, we detected full-length β3 subunit trimers on the plasma membrane of transfected HEK293 cells. We further show that β3 subunits can bind to more than one site on the Na_v 1.5 α subunit and induce the formation of α subunit oligomers, including trimers. Our results suggest a new and unexpected role for the β3 subunits in Na_v channel cross-linking and provide new structural insights into some pathological Na_v channel mutations.     

Crystal Structure of Calmodulin Binding Domain of Orai1 in Complex with Ca2+?Calmodulin Displays a Unique Binding Mode

Orai1 is a plasma membrane protein that in its tetrameric form is responsible for calcium influx from the extracellular environment into the cytosol in response to interaction with the Ca²⁺-depletion sensor STIM1. This is followed by a fast Ca²⁺·calmodulin (CaM)-dependent inhibition, resulting from CaM binding to an Orai1 region called the calmodulin binding domain (CMBD). The interaction between Orai1 and CaM at the atomic level remains unknown. Here, we report the crystal structure of a CaM·Orai1-CMBD complex showing one CMBD bound to the C-terminal lobe of CaM, differing from other CaM-target protein complexes, in which both N- and C-terminal lobes of CaM (CaM-N and CaM-C) are involved in target binding. Orai1-CMBD binds CaM-C mainly through hydrophobic interactions, primarily involving residue Trp⁷⁶ of Orai1-CMBD, which interacts with the hydrophobic pocket of CaM-C. However, NMR data, isothermal titration calorimetry data, and pulldown assays indicated that CaM-N and CaM-C both can bind Orai1-CMBD, with CaM-N having ∼4 times weaker affinity than CaM-C. Pulldown assays of a Orai1-CMBD(W76E) mutant, gel filtration chromatography data, and NOE signals indicated that CaM-N and CaM-C can each bind one Orai1-CMBD. Thus our studies support an unusual, extended 1:2 binding mode of CaM to Orai1-CMBDs, and quantify the affinity of Orai1 for CaM. We propose a two-step mechanism for CaM-dependent Orai1 inactivation initiated by binding of the C-lobe of CaM to the CMBD of one Orai1 followed by the binding of the N-lobe of CaM to the CMBD of a neighboring Orai1.     

Crystal Structure of BamB Bound to a Periplasmic Domain Fragment of BamA,the Central Component of the β-Barrel Assembly Machine

The β-barrel assembly machinery (BAM) mediates folding and insertion of β-barrel outer membrane proteins (OMPs) into the outer membrane of Gram-negative bacteria. BAM is a five-protein complex consisting of the β-barrel OMP BamA and lipoproteins BamB, -C, -D, and -E. High resolution structures of all the individual BAM subunits and a BamD-BamC complex have been determined. However, the overall complex architecture remains elusive. BamA is the central component of BAM and consists of a membrane-embedded β-barrel and a periplasmic domain with five polypeptide translocation-associated (POTRA) motifs thought to interact with the accessory lipoproteins. Here we report the crystal structure of a fusion between BamB and a POTRA3–5 fragment of BamA. Extended loops 13 and 17 protruding from one end of the BamB β-propeller contact the face of the POTRA3 β-sheet in BamA. The interface is stabilized by several hydrophobic contacts, a network of hydrogen bonds, and a cation-π interaction between BamA Tyr-255 and BamB Arg-195. Disruption of BamA-BamB binding by BamA Y255A and probing of the interface by disulfide bond cross-linking validate the physiological relevance of the observed interface. Furthermore, the structure is consistent with previously published mutagenesis studies. The periplasmic five-POTRA domain of BamA is flexible in solution due to hinge motions in the POTRA2–3 linker. Modeling BamB in complex with full-length BamA shows BamB binding at the POTRA2–3 hinge, suggesting a role in modulation of BamA flexibility and the conformational changes associated with OMP folding and insertion.     

Molecular Mechanism of Thioflavin-T Binding to the Surface of β-Rich Peptide Self-Assemblies

A number of small organic molecules have been developed that bind to amyloid fibrils, a subset of which also inhibit fibrillization. Among these, the benzothiol dye Thioflavin-T (ThT) has been used for decades in the diagnosis of protein-misfolding diseases and in kinetic studies of self-assembly (fibrillization). Despite its importance, efforts to characterize the ThT-binding mechanism at the atomic level have been hampered by the inherent insolubility and heterogeneity of peptide self-assemblies. To overcome these challenges, we have developed a minimalist approach to designing a ThT-binding site in a "peptide self-assembly mimic” (PSAM) scaffold. PSAMs are engineered water-soluble proteins that mimic a segment of β-rich peptide self-assembly, and they are amenable to standard biophysical techniques and systematic mutagenesis. The PSAM β-sheet contains rows of repetitive amino acid patterns running perpendicular to the strands (cross-strand ladders) that represent a ubiquitous structural feature of fibril-like surfaces. We successfully designed a ThT-binding site that recapitulates the hallmarks of ThT-fibril interactions by constructing a cross-strand ladder consisting of contiguous tyrosines. The X-ray crystal structures suggest that ThT interacts with the β-sheet by docking onto surfaces formed by a single tyrosine ladder, rather than in the space between adjacent ladders. Systematic mutagenesis further demonstrated that tyrosine surfaces across four or more β-strands formed the minimal binding site for ThT. Our work thus provides structural insights into how this widely used dye recognizes a prominent subset of peptide self-assemblies, and proposes a strategy to elucidate the mechanisms of fibril-ligand interactions.     

Insights into the Molecular Mechanisms of CO2-Mediated Regulation of Stomatal Movements

Plants must continually balance the influx of CO₂ for photosynthesis against the loss of water vapor through stomatal pores in their leaves. This balance can be achieved by controlling the aperture of the stomatal pores in response to several environmental stimuli. Elevation in atmospheric [CO₂] induces stomatal closure and further impacts leaf temperatures, plant growth and water-use efficiency, and global crop productivity. Here, we review recent advances in understanding CO₂-perception mechanisms and CO₂-mediated signal transduction in the regulation of stomatal movements, and we explore how these mechanisms are integrated with other signaling pathways in guard cells.     

Crystal Structures of Human CaMKIα Reveal Insights into the Regulation Mechanism of CaMKI

Human calcium/calmodulin-dependent protein kinase I (CaMKI) plays pivotal roles in the nervous system. The activity of human CaMKI is regulated by a regulatory region including an autoinhibitory segment and a CaM-binding segment. We report here four structures of three CaMKIα truncates in apo form and in complexes with ATP. In an apo, autoinhibited structure, the activation segment adopts a unique helical conformation which together with the autoinhibitory segment constrains helices αC and αD in inactive conformations, sequesters Thr177 from being phosphorylated, and occludes the substrate-binding site. In an ATP-bound, inactive structure, the activation segment is largely disordered and the CaM-binding segment protrudes out ready for CaM binding. In an ATP-bound, active structure, the regulatory region is dissociated from the catalytic core and the catalytic site assumes an active conformation. Detailed structural analyses reveal the interplay of the regulatory region, the activation segment, and the nucleotide-binding site in the regulation of CaMKI.     

A Crystal Structure of the Cyclic GMP-dependent Protein Kinase Iβ Dimerization/Docking Domain Reveals Molecular Details of Isoform-specific Anchoring

Cyclic GMP-dependent protein kinase (PKG) is a key mediator of the nitric oxide/cGMP signaling pathway and plays a central role in regulating cardiovascular and neuronal functions. The N-terminal ∼50 amino acids of the kinase are required for homodimerization and association with isoform-specific PKG-anchoring proteins (GKAPs), which target the kinase to specific substrates. To understand the molecular details of PKG dimerization and gain insight into its association with GKAPs, we solved a crystal structure of the PKG Iβ dimerization/docking domain. Our structure provides molecular details of this unique leucine/isoleucine zipper, revealing specific hydrophobic and ionic interactions that mediate dimerization and demonstrating the topology of the GKAP interaction surface.     

Crystal Structures of an Oligopeptide-Binding Protein from the Biosynthetic Pathway of the β-Lactamase Inhibitor Clavulanic Acid

Clavulanic acid (CA) is a clinically important β-lactamase inhibitor that is produced by fermentation of Streptomyces clavuligerus. The CA biosynthesis pathway starts from arginine and glyceraldehyde-3-phosphate and proceeds via (3S,5S)-clavaminic acid, which is converted to (3R,5R)-clavaldehyde, the immediate precursor of (3R,5R)-CA. Open reading frames 7 (orf7) and 15 (orf15) of the CA biosynthesis cluster encode oligopeptide-binding proteins (OppA1 and OppA2), which are essential for CA biosynthesis. OppA1/2 are proposed to be involved in the binding and/or transport of peptides across the S. clavuligerus cell membrane. Peptide binding assays reveal that recombinant OppA1 and OppA2 bind di-/tripeptides containing arginine and certain nonapeptides including bradykinin. Crystal structures of OppA2 in its apo form and in complex with arginine or bradykinin were solved to 1.45, 1.7, and 1.7 Å resolution, respectively. The overall fold of OppA2 consists of two lobes with a deep cavity in the center, as observed for other oligopeptide-binding proteins. The large cavity creates a peptide/arginine binding cleft. The crystal structures of OppA2 in complex with arginine or bradykinin reveal that the C-terminal arginine of bradykinin binds similarly to arginine. The results are discussed in terms of the possible roles of OppA1/2 in CA biosynthesis.     

The Structure of the T190M Mutant of Murine α-Dystroglycan at High Resolution: Insight into the Molecular Basis of a Primary Dystroglycanopathy

The severe dystroglycanopathy known as a form of limb-girdle muscular dystrophy (LGMD2P) is an autosomal recessive disease caused by the point mutation T192M in α-dystroglycan. Functional expression analysis in vitro and in vivo indicated that the mutation was responsible for a decrease in posttranslational glycosylation of dystroglycan, eventually interfering with its extracellular-matrix receptor function and laminin binding in skeletal muscle and brain. The X-ray crystal structure of the missense variant T190M of the murine N-terminal domain of α-dystroglycan (50-313) has been determined, and showed an overall topology (Ig-like domain followed by a basket-shaped domain reminiscent of the small subunit ribosomal protein S6) very similar to that of the wild-type structure. The crystallographic analysis revealed a change of the conformation assumed by the highly flexible loop encompassing residues 159–180. Moreover, a solvent shell reorganization around Met190 affects the interaction between the B1–B5 anti-parallel strands forming part of the floor of the basket-shaped domain, with likely repercussions on the folding stability of the protein domain(s) and on the overall molecular flexibility. Chemical denaturation and limited proteolysis experiments point to a decreased stability of the T190M variant with respect to its wild-type counterpart. This mutation may render the entire L-shaped protein architecture less flexible. The overall reduced flexibility and stability may affect the functional properties of α-dystroglycan via negatively influencing its binding behavior to factors needed for dystroglycan maturation, and may lay the molecular basis of the T190M-driven primary dystroglycanopathy.     

Structure,Function, and Dynamics of the Gα Binding Domain of Ric-8A

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The 1.7 ? X-Ray Crystal Structure of the Porcine Factor VIII C2 Domain and Binding Analysis to Anti-Human C2 Domain Antibodies and Phospholipid Surfaces

The factor VIII C2 domain is essential for binding to activated platelet surfaces as well as the cofactor activity of factor VIII in blood coagulation. Inhibitory antibodies against the C2 domain commonly develop following factor VIII replacement therapy for hemophilia A patients, or they may spontaneously arise in cases of acquired hemophilia. Porcine factor VIII is an effective therapeutic for hemophilia patients with inhibitor due to its low cross-reactivity; however, the molecular basis for this behavior is poorly understood. In this study, the X-ray crystal structure of the porcine factor VIII C2 domain was determined, and superposition of the human and porcine C2 domains demonstrates that most surface-exposed differences cluster on the face harboring the “non-classical” antibody epitopes. Furthermore, antibody-binding results illustrate that the “classical” 3E6 antibody can bind both the human and porcine C2 domains, although the inhibitory titer to human factor VIII is 41 Bethesda Units (BU)/mg IgG versus 0.8 BU/mg IgG to porcine factor VIII, while the non-classical G99 antibody does not bind to the porcine C2 domain nor inhibit porcine factor VIII activity. Further structural analysis of differences between the electrostatic surface potentials suggest that the C2 domain binds to the negatively charged phospholipid surfaces of activated platelets primarily through the 3E6 epitope region. In contrast, the G99 face, which contains residue 2227, should be distal to the membrane surface. Phospholipid binding assays indicate that both porcine and human factor VIII C2 domains bind with comparable affinities, and the human K2227A and K2227E mutants bind to phospholipid surfaces with similar affinities as well. Lastly, the G99 IgG bound to PS-immobilized factor VIII C2 domain with an apparent dissociation constant of 15.5 nM, whereas 3E6 antibody binding to PS-bound C2 domain was not observed.     

Inhibition of αIIbβ3 Ligand Binding by an αIIb Peptide that Clasps the Hybrid Domain to the βI Domain of β3

Agonist-stimulated platelet activation triggers conformational changes of integrin αIIbβ3, allowing fibrinogen binding and platelet aggregation. We have previously shown that an octapeptide, ^p1YMESRADR⁸, corresponding to amino acids 313–320 of the β-ribbon extending from the β-propeller domain of αIIb, acts as a potent inhibitor of platelet aggregation. Here we have performed in silico modelling analysis of the interaction of this peptide with αIIbβ3 in its bent and closed (not swing-out) conformation and show that the peptide is able to act as a substitute for the β-ribbon by forming a clasp restraining the β3 hybrid and βI domains in a closed conformation. The involvement of species-specific residues of the β3 hybrid domain (E356 and K384) and the β1 domain (E297) as well as an intrapeptide bond (^pE315-^pR317) were confirmed as important for this interaction by mutagenesis studies of αIIbβ3 expressed in CHO cells and native or substituted peptide inhibitory studies on platelet functions. Furthermore, NMR data corroborate the above results. Our findings provide insight into the important functional role of the αIIb β-ribbon in preventing integrin αIIbβ3 head piece opening, and highlight a potential new therapeutic approach to prevent integrin ligand binding.     

Crystal Structure of the Human Pol α B Subunit in Complex with the C-terminal Domain of the Catalytic Subunit

In eukaryotic DNA replication, short RNA-DNA hybrid primers synthesized by primase-DNA polymerase α (Prim-Pol α) are needed to start DNA replication by the replicative DNA polymerases, Pol δ and Pol ϵ. The C terminus of the Pol α catalytic subunit (p180_C) in complex with the B subunit (p70) regulates the RNA priming and DNA polymerizing activities of Prim-Pol α. It tethers Pol α and primase, facilitating RNA primer handover from primase to Pol α. To understand these regulatory mechanisms and to reveal the details of human Pol α organization, we determined the crystal structure of p70 in complex with p180_C. The structured portion of p70 includes a phosphodiesterase (PDE) domain and an oligonucleotide/oligosaccharide binding (OB) domain. The N-terminal domain and the linker connecting it to the PDE domain are disordered in the reported crystal structure. The p180_C adopts an elongated asymmetric saddle shape, with a three-helix bundle in the middle and zinc-binding modules (Zn1 and Zn2) on each side. The extensive p180_C-p70 interactions involve 20 hydrogen bonds and a number of hydrophobic interactions resulting in an extended buried surface of 4080 Ų. Importantly, in the structure of the p180_C-p70 complex with full-length p70, the residues from the N-terminal to the OB domain contribute to interactions with p180_C. The comparative structural analysis revealed both the conserved features and the differences between the human and yeast Pol α complexes.