Protein Structure and Folding
Structural analysis of the interaction between human cytokine BMP-2 and the antagonist Noggin reveals molecular details of cell chondrogenesis inhibitionBone morphogenetic proteins (BMPs) are secreted cytokines belonging to the transforming growth factor-β superfamily. New therapeutic approaches based on BMP activity, particularly for cartilage and bone repair, have sparked considerable interest; however, a lack of understanding of their interaction pathways and the side effects associated with their use as biopharmaceuticals have dampened initial enthusiasm. Here, we used BMP-2 as a model system to gain further insight into both the relationship between structure and function in BMPs and the principles that govern affinity for their cognate antagonist Noggin.
A seven-residue deletion in PrP leads to generation of a spontaneous prion formed from C-terminal C1 fragment of PrPPrions result from a drastic conformational change of the host-encoded cellular prion protein (PrP), leading to the formation of β-sheet–rich, insoluble, and protease-resistant self-replicating assemblies (PrPSc). The cellular and molecular mechanisms involved in spontaneous prion formation in sporadic and inherited human prion diseases or equivalent animal diseases are poorly understood, in part because cell models of spontaneously forming prions are currently lacking. Here, extending studies on the role of the H2 α-helix C terminus of PrP, we found that deletion of the highly conserved 190HTVTTTT196 segment of ovine PrP led to spontaneous prion formation in the RK13 rabbit kidney cell model.
Human cytomegalovirus-specific T-cell receptor engineered for high affinity and soluble expression using mammalian cell displayT-cell receptors (TCR) have considerable potential as therapeutics and antibody-like reagents to monitor disease progression and vaccine efficacy. Whereas antibodies recognize only secreted and surface-bound proteins, TCRs recognize otherwise inaccessible disease-associated intracellular proteins when they are presented as processed peptides bound to major histocompatibility complexes (pMHC). TCRs have been primarily explored for cancer therapy applications but could also target infectious diseases such as cytomegalovirus (CMV).
Crystal structures of the human 4-1BB receptor bound to its ligand 4-1BBL reveal covalent receptor dimerization as a potential signaling amplifierHuman (h)4-1BB (TNFRSF9 or CD137) is an inducible tumor necrosis factor receptor (TNFR) superfamily member that interacts with its cognate ligand h4-1BBL to promote T lymphocyte activation and proliferation. h4-1BB is currently being targeted with agonists in cancer immunotherapy. Here, we determined the crystal structures of unbound h4-1BBL and both WT h4-1BB and a dimerization-deficient h4-1BB mutant (C121S) in complex with h4-1BBL at resolutions between 2.7 and 3.2 Å. We observed that the structural arrangement of 4-1BBL, both unbound and in the complex, represents the canonical bell shape as seen in other similar TNF proteins and differs from the previously reported three-bladed propeller structure of 4-1BBL.
Crystal structure of murine 4-1BB and its interaction with 4-1BBL support a role for galectin-9 in 4-1BB signaling4-1BB (CD137) is a TNF receptor superfamily (TNFRSF) member that is thought to undergo receptor trimerization upon binding to its trimeric TNF superfamily ligand (4-1BBL) to stimulate immune responses. 4-1BB also can bind to the tandem repeat–type lectin galectin-9 (Gal-9), and signaling through mouse (m)4-1BB is reduced in galectin-9 (Gal-9)–deficient mice, suggesting a pivotal role of Gal-9 in m4-1BB activation. Here, using sulfur-SAD phasing, we determined the crystal structure of m4-1BB to 2.2-Å resolution.
The crystal structure of full-length Sizzled from Xenopus laevis yields insights into Wnt-antagonistic function of secreted Frizzled-related proteinsThe Wnt-signaling pathway is crucial to cell proliferation, differentiation, and migration. The secreted Frizzled-related proteins (sFRPs) represent the largest family of secreted Wnt inhibitors. However, their function in antagonizing Wnt signaling has remained somewhat controversial. Here, we report the crystal structure of Sizzled from Xenopus laevis, the first full-length structure of an sFRP. Tethered by an inter-domain disulfide bond and a linker, the N-terminal cysteine-rich domain (CRD) and the C-terminal netrin-like domain (NTR) of Sizzled are arranged in a tandem fashion, with the NTR domain occluding the groove of CRD for Wnt accessibility.
A structural organization for the Disrupted in Schizophrenia 1 protein, identified by high-throughput screening, reveals distinctly folded regions, which are bisected by mental illness-related mutationsDisrupted in Schizophrenia 1 (DISC1) is a scaffolding protein of significant importance for neurodevelopment and a prominent candidate protein in the pathology of major mental illness. DISC1 modulates a number of critical neuronal signaling pathways through protein-protein interactions; however, the mechanism by which this occurs and how DISC1 causes mental illness is unclear, partly because knowledge of the structure of DISC1 is lacking. A lack of homology with known proteins has hindered attempts to define its domain composition.
Effector-attenuating Substitutions That Maintain Antibody Stability and Reduce Toxicity in MiceThe antibody Fc region regulates antibody cytotoxic activities and serum half-life. In a therapeutic context, however, the cytotoxic effector function of an antibody is often not desirable and can create safety liabilities by activating native host immune defenses against cells expressing the receptor antigens. Several amino acid changes in the Fc region have been reported to silence or reduce the effector function of antibodies. These earlier studies focused primarily on the interaction of human antibodies with human Fc-γ receptors, and it remains largely unknown how such changes to Fc might translate to the context of a murine antibody.
Consequences of Glycine Mutations in the Fibronectin-binding Sequence of CollagenCollagen and fibronectin (Fn) are two key extracellular matrix proteins, which are known to interact and jointly shape matrix structure and function. Most proteins that interact with collagen bind only to the native triple-helical form, whereas Fn is unusual in binding strongly to denatured collagen and more weakly to native collagen. The consequences of replacing a Gly by Ser at each position in the required (Gly-Xaa-Yaa)6 Fn-binding sequence are probed here, using model peptides and a recombinant bacterial collagen system.
Structural and Immunological Characterization of Recombinant 6-Cysteine Domains of the Plasmodium falciparum Sexual Stage Protein Pfs230Development of a Plasmodium falciparum (Pf) transmission blocking vaccine (TBV) has the potential to significantly impact malaria control. Antibodies elicited against sexual stage proteins in the human bloodstream are taken up with the blood meal of the mosquitoes and inactivate parasite development in the mosquito. In a phase 1 trial, a leading TBV identified as Pfs25-EPA/Alhydrogel® appeared safe and immunogenic, however, the level of Pfs25-specific antibodies were likely too low for an effective vaccine.
Functional Human α7 Nicotinic Acetylcholine Receptor (nAChR) Generated from Escherichia coliHuman Cys-loop receptors are important therapeutic targets. High-resolution structures are essential for rational drug design, but only a few are available due to difficulties in obtaining sufficient quantities of protein suitable for structural studies. Although expression of proteins in E. coli offers advantages of high yield, low cost, and fast turnover, this approach has not been thoroughly explored for full-length human Cys-loop receptors because of the conventional wisdom that E. coli lacks the specific chaperones and post-translational modifications potentially required for expression of human Cys-loop receptors.
Recombinant Collagen Engineered to Bind to Discoidin Domain Receptor Functions as a Receptor InhibitorA bacterial collagen-like protein Scl2 has been developed as a recombinant collagen model system to host human collagen ligand-binding sequences, with the goal of generating biomaterials with selective collagen bioactivities. Defined binding sites in human collagen for integrins, fibronectin, heparin, and MMP-1 have been introduced into the triple-helical domain of the bacterial collagen and led to the expected biological activities. The modular insertion of activities is extended here to the discoidin domain receptors (DDRs), which are collagen-activated receptor tyrosine kinases.
Optimal Neutralization of Centruroides noxius Venom Is Understood through a Structural Complex between Two Antibody Fragments and the Cn2 ToxinThe current trend of using recombinant antibody fragments in research to develop novel antidotes against scorpion stings has achieved excellent results. The polyclonal character of commercial antivenoms, obtained through the immunization of animals and which contain several neutralizing antibodies that recognize different epitopes on the toxins, guarantees the neutralization of the venoms. To avoid the use of animals, we aimed to develop an equivalent recombinant antivenom composed of a few neutralizing single chain antibody fragments (scFvs) that bind to two different epitopes on the scorpion toxins.
A New Approach to Produce HIV-1 Envelope Trimers: BOTH CLEAVAGE AND PROPER GLYCOSYLATION ARE ESSENTIAL TO GENERATE AUTHENTIC TRIMERSBackground: HIV-1 envelope trimer is a candidate for designing an effective HIV vaccine.Results: gp140 attached to Strep-tag through a long linker is used to purify HIV trimers. Cleaved, uncleaved, and fully and partially glycosylated trimers are characterized.Conclusion: Cleaved and glycosylated gp140 assembles into authentic propeller-shaped trimers.Significance: This system could generate HIV-1 trimers for clinical trials and vaccine manufacture.