Pavel Strop

Transglutaminase 2 undergoes a large conformational change upon activation

Human transglutaminase 2 (TG2), a member of a large family of enzymes that catalyze protein crosslinking, plays an important role in the extracellular matrix biology of many tissues and is implicated in the gluten-induced pathogenesis of celiac sprue. Although vertebrate transglutaminases have been studied extensively, thus far all structurally characterized members of this family have been crystallized in conformations with inaccessible active sites. We have trapped human TG2 in complex with an inhibitor that mimics inflammatory gluten peptide substrates and have solved, at 2-Å resolution, its x-ray crystal structure. The inhibitor stabilizes TG2 in an extended conformation that is dramatically different from earlier transglutaminase structures. The active site is exposed, revealing that catalysis takes place in a tunnel, bridged by two tryptophan residues that separate acyl-donor from acyl-acceptor and stabilize the tetrahedral reaction intermediates. Site-directed mutagenesis was used to investigate the acyl-acceptor side of the tunnel, yielding mutants with a marked increase in preference for hydrolysis over transamidation. By providing the ability to visualize this activated conformer, our results create a foundation for understanding the catalytic as well as the non-catalytic roles of TG2 in biology, and for dissecting the process by which the autoantibody response to TG2 is induced in celiac sprue patients.

Structures of the Prokaryotic Mechanosensitive Channels MscL and MscS

Publisher Summary This chapter describes the crystallographic analyses of the Mycobacterium tuberculosis mechanosensitive channels of large (MscL) and the Escherichia coli mechanosensitive channels of small (MscS). Crystal structures of the M. tuberculosis MscL and E. coli MscS were initially reported at 3.5‐ and 3.9‐ A resolutions, respectively. The basic structural framework of the MscL and MscS transmembrane domains is provided by α ‐helices; each subunit of MscL has two helices for a total of 10, whereas MscS has three helices per subunit for a total of 21. From a structural perspective, MscL and MscS represent fascinating targets as they provide an opportunity to explore the coupling between protein conformation and the membrane environment responsible for channel gating. Tension and pressure sensitive systems, such as MscL and MscS, have the attraction that these environmental properties are energetically coupled to changes in protein area and volume, respectively that may be directly quantitated from structural models.

Single-molecule FRET–derived model of the synaptotagmin 1–SNARE fusion complex

Synchronous neurotransmission is triggered when Ca2+ binds to synaptotagmin 1 (Syt1), a synaptic-vesicle protein that interacts with SNAREs and membranes. We used single-molecule fluorescence resonance energy transfer (FRET) between synaptotagmins two C2 domains to determine that their conformation consists of multiple states with occasional transitions, consistent with domains in random relative motion. SNARE binding results in narrower intrasynaptotagmin FRET distributions and less frequent transitions between states. We obtained an experimentally determined model of the elusive Syt1–SNARE complex using a multibody docking approach with 34 FRET-derived distances as restraints. The Ca2+-binding loops point away from the SNARE complex, so they may interact with the same membrane. The loop arrangement is similar to that of the crystal structure of SNARE-induced Ca2+-bound Syt3, suggesting a common mechanism by which the interaction between synaptotagmins and SNAREs aids in Ca2+-triggered fusion.

Structures of Neuroligin-1 and the Neuroligin-1/Neurexin-1β Complex Reveal Specific Protein-Protein and Protein-Ca2+ Interactions

Neurexins and neuroligins provide trans-synaptic connectivity by the Ca2+-dependent interaction of their alternatively spliced extracellular domains. Neuroligins specify synapses in an activity-dependent manner, presumably by binding to neurexins. Here, we present the crystal structures of neuroligin-1 in isolation and in complex with neurexin-1 beta. Neuroligin-1 forms a constitutive dimer, and two neurexin-1 beta monomers bind to two identical surfaces on the opposite faces of the neuroligin-1 dimer to form a heterotetramer. The neuroligin-1/neurexin-1 beta complex exhibits a nanomolar affinity and includes a large binding interface that contains bound Ca2+. Alternatively spliced sites in neurexin-1 beta and in neuroligin-1 are positioned nearby the binding interface, explaining how they regulate the interaction. Structure-based mutations of neuroligin-1 at the interface disrupt binding to neurexin-1 beta, but not the folding of neuroligin-1 and confirm the validity of the binding interface of the neuroligin-1/neurexin-1 beta complex. Our results provide molecular insights for understanding the role of cell-adhesion proteins in synapse function.

Refractive index-based determination of detergent concentration and its application to the study of membrane proteins

The concentration of detergent in membrane protein preparations can have a critical role on protein stability, function, and the potential for crystallization. Unfortunately, dialysis or protein concentration can lead to an unknown amount of detergent in the final membrane protein preparations. Here we present a method for the determination of detergent concentration based on refractive index of the detergent solution. This method was applied to quantitate the amount of detergent remaining in solution after concentration in various concentrators. We found that the ability of the tested detergents to pass through the molecular weight cutoff membrane correlates well with detergent micelle size. Therefore, the micelle size can be used as a rough guide to estimate the retention of a given detergent in various molecular weight cutoff concentrators. The refractive index method is exceptionally informative when coupled with size exclusion chromatography and light scattering, and can be used to determine the oligomeric state of the membrane protein, the size of a protein‐associated micelle, as well as the amount and size of the unbound detergent micelle.

Increasing Serum Half-life and Extending Cholesterol Lowering in Vivo by Engineering Antibody with pH-sensitive Binding to PCSK9

Background: An antagonistic anti-PCSK9 antibody exhibits target-mediated clearance, resulting in a dose-dependent PK. Results: Engineering of an antibody with pH-sensitive binding to PCSK9 decreases target-mediated clearance, resulting in increased PK and efficacy in vivo. Conclusion: pH-sensitive anti-PCSK9 antibodies are excellent candidates for therapeutic development. Significance: pH-sensitive antibodies may enable less frequent or lower dosing of antibodies hampered by target-mediated clearance and high antigen load. Target-mediated clearance and high antigen load can hamper the efficacy and dosage of many antibodies. We show for the first time that the mouse, cynomolgus, and human cross-reactive, antagonistic anti-proprotein convertase substilisin kexin type 9 (PCSK9) antibodies J10 and the affinity-matured and humanized J16 exhibit target-mediated clearance, resulting in dose-dependent pharmacokinetic profiles. These antibodies prevent the degradation of low density lipoprotein receptor, thus lowering serum levels of LDL-cholesterol and potently reducing serum cholesterol in mice, and selectively reduce LDL-cholesterol in cynomolgus monkeys. In order to increase the pharmacokinetic and efficacy of this promising therapeutic for hypercholesterolemia, we engineered pH-sensitive binding to mouse, cynomolgus, and human PCSK9 into J16, resulting in J17. This antibody shows prolonged half-life and increased duration of cholesterol lowering in two species in vivo by binding to endogenous PCSK9 in mice and cynomolgus monkeys, respectively. The proposed mechanism of this pH-sensitive antibody is that it binds with high affinity to PCSK9 in the plasma at pH 7.4, whereas the antibody-antigen complex dissociates at the endosomal pH of 5.5–6.0 in order to escape from target-mediated degradation. Additionally, this enables the antibody to bind to another PCSK9 and therefore increase the antigen-binding cycles. Furthermore, we show that this effect is dependent on the neonatal Fc receptor, which rescues the dissociated antibody in the endosome from degradation. Engineered pH-sensitive antibodies may enable less frequent or lower dosing of antibodies hampered by target-mediated clearance and high antigen load.

Proprotein Convertase Substilisin/Kexin Type 9 Antagonism Reduces Low-Density Lipoprotein Cholesterol in Statin-Treated Hypercholesterolemic Nonhuman Primates

Proprotein convertase substilisin/kexin type 9 (PCSK9) promotes the degradation of low-density lipoprotein (LDL) receptor (LDLR) and thereby increases serum LDL-cholesterol (LDL-C). We have developed a humanized monoclonal antibody that recognizes the LDLR binding domain of PCSK9. This antibody, J16, and its precursor mouse antibody, J10, potently inhibit PCSK9 binding to the LDLR extracellular domain and PCSK9-mediated down-regulation of LDLR in vitro. In vivo, J10 effectively reduces serum cholesterol in C57BL/6 mice fed normal chow. J16 reduces LDL-C in healthy and diet-induced hypercholesterolemic cynomologous monkeys, but does not significantly affect high-density lipoprotein-cholesterol. Furthermore, J16 greatly lowered LDL-C in hypercholesterolemic monkeys treated with the HMG-CoA reductase inhibitor simvastatin. Our data demonstrate that anti-PCSK9 antibody is a promising LDL-C-lowering agent that is both efficacious and potentially additive to current therapies.

Molecular mechanism of the synaptotagmin–SNARE interaction in Ca2+-triggered vesicle fusion

In neurons, SNAREs, synaptotagmin and other factors catalyze Ca2+-triggered fusion of vesicles with the plasma membrane. The molecular mechanism of this process, especially the interaction between synaptotagmin and SNAREs, remains an enigma. Here we characterized this interaction by single-molecule fluorescence microscopy and crystallography. The two rigid Ca2+-binding domains of synaptotagmin 3 (Syt3) undergo large relative motions in solution. Interaction with SNARE complex amplifies a particular state of the two domains that is further enhanced by Ca2+. This state is represented by the first SNARE-induced Ca2+-bound crystal structure of a synaptotagmin fragment containing both domains. The arrangement of the Ca2+-binding loops of this structure of Syt3 matches that of SNARE-bound Syt1, suggesting a conserved feature of synaptotagmins. The loops resemble the membrane-interacting loops of certain viral fusion proteins in the postfusion state, suggesting unexpected similarities between both fusion systems.

Three-dimensional molecular modeling with single molecule FRET.

Single molecule fluorescence energy transfer experiments enable investigations of macromolecular conformation and folding by the introduction of fluorescent dyes at specific sites in the macromolecule. Multiple such experiments can be performed with different labeling site combinations in order to map complex conformational changes or interactions between multiple molecules. Distances that are derived from such experiments can be used for determination of the fluorophore positions by triangulation. When combined with a known structure of the macromolecule(s) to which the fluorophores are attached, a three-dimensional model of the system can be determined. However, care has to be taken to properly derive distance from fluorescence energy transfer efficiency and to recognize the systematic or random errors for this relationship. Here we review the experimental and computational methods used for three-dimensional modeling based on single molecule fluorescence resonance transfer, and describe recent progress in pushing the limits of this approach to macromolecular complexes.

An autonomous CDR3δ is sufficient for recognition of the nonclassical MHC class I molecules T10 and T22 by γδ T cells

It remains unclear whether γδ T cell receptors (TCRs) detect antigens in a manner similar to antibodies or αβ TCRs. Here we show that reactivity between G8 and KN6 γδ TCRs and the MHC class Ib molecule T22 can be transplanted, with retention of wild-type ligand affinity, after en bloc grafting of G8 and KN6 CDR3δ loops in place onto the CDR3α loop of an αβ TCR. We also find that a shared sequence motif within CDR3δ loops of all T22-reactive γδ TCRs binds T22 in energetically distinct fashions, and that T10d, which binds G8 with weak affinity, is converted into a high-affinity ligand by a single point mutation. These results demonstrate an unprecedented autonomy of a single CDR3 loop in antigen recognition.It remains unclear whether γδ T cell antigen receptors (TCRs) detect antigens in a way similar to antibodies or αβ TCRs. Here we show that reactivity between the G8 and KN6 γδ TCRs and the major histocompatibility complex class Ib molecule T22 could be recapitulated, with retention of wild-type ligand affinity, in an αβ TCR after grafting of a G8 or KN6 complementarity-determining region 3-δ (CDR3δ) loop in place of the CDR3α loop of an αβ TCR. We also found that a shared sequence motif in CDR3δ loops of all T22-reactive γδ TCRs bound T22 in energetically distinct ways, and that T10d, which bound G8 with weak affinity, was converted into a high-affinity ligand by a single point mutation. Our results demonstrate unprecedented autonomy of a single CDR3 loop in antigen recognition.