Multistage protective anti-CelTOS monoclonal antibodies with cross-species sterile protection against malaria.

CelTOS is a malaria vaccine antigen that is conserved in Plasmodium and other apicomplexan parasites and plays a role in cell-traversal. The structural basis and mechanisms of CelTOS-induced protective immunity to parasites are unknown. Here, CelTOS-specific monoclonal antibodies (mAbs) 7g7 and 4h12 demonstrated multistage activity, protecting against liver infection and preventing parasite transmission to mosquitoes.

Conformations of Bcs1L undergoing ATP hydrolysis suggest a concerted translocation mechanism for folded iron-sulfur protein substrate.

The human AAA-ATPase Bcs1L translocates the fully assembled Rieske iron-sulfur protein (ISP) precursor across the mitochondrial inner membrane, enabling respiratory Complex III assembly. Exactly how the folded substrate is bound to and released from Bcs1L has been unclear, and there has been ongoing debate as to whether subunits of Bcs1L act in sequence or in unison hydrolyzing ATP when moving the protein cargo. Here, we captured Bcs1L conformations by cryo-EM during active ATP hydrolysis in the presence or absence of ISP substrate.

Sequence-structure-function characterization of the emerging tetracycline destructase family of antibiotic resistance enzymes.

Tetracycline destructases (TDases) are flavin monooxygenases which can confer resistance to all generations of tetracycline antibiotics. The recent increase in the number and diversity of reported TDase sequences enables a deep investigation of the TDase sequence-structure-function landscape. Here, we evaluate the sequence determinants of TDase function through two complementary approaches: (1) constructing profile hidden Markov models to predict new TDases, and (2) using multiple sequence alignments to identify conserved positions important to protein function.

A human antibody epitope map of the malaria vaccine antigen Pfs25.

Pfs25 is a leading antigen for a malaria transmission-blocking vaccine and shows moderate transmission-blocking activity and induction of rapidly decreasing antibody titers in clinical trials. A comprehensive definition of all transmission-reducing epitopes of Pfs25 will inform structure-guided design to enhance Pfs25-based vaccines, leading to potent transmission-blocking activity. Here, we compiled a detailed human antibody epitope map comprising epitope binning data and structures of multiple human monoclonal antibodies, including three new crystal structures of Pfs25 in complex with transmission-reducing antibodies from Malian volunteers immunized with Pfs25 conjugated to EPA and adjuvanted with AS01.

Structures of Cancer Antigen Mesothelin and Its Complexes with Therapeutic Antibodies.

Unlabelled: The tumor-associated antigen mesothelin is expressed at high levels on the cell surface of many human cancers, while its expression in normal tissues is limited. The binding of mesothelin to the tumor-associated cancer antigen 125 (CA-125) can lead to heterotypic cell adhesion and tumor metastasis within the pleural and peritoneal cavities. Immunotherapeutic strategies targeting mesothelin are being intensively investigated.

A human antibody epitope map of Pfs230D1 derived from analysis of individuals vaccinated with a malaria transmission-blocking vaccine.

Pfs230 domain 1 (Pfs230D1) is an advanced malaria transmission-blocking vaccine antigen demonstrating high functional activity in clinical trials. However, the structural and functional correlates of transmission-blocking activity are not defined. Here, we characterized a panel of human monoclonal antibodies (hmAbs) elicited in vaccinees immunized with Pfs230D1.

Structural analysis of Plasmodium falciparum ookinete surface antigen Pfs28 relevant for malaria vaccine design.

Pfs28 is a Plasmodium falciparum malaria transmission-blocking vaccine candidate that is anchored to the parasite surface through a C-terminal glycosylphosphatidylinositol (GPI) moiety, and plays a role in parasite survival in the mosquito midgut. Pfs28 contains epidermal growth factor (EGF)-like domains and is part of a family of sexual stage malaria proteins that includes the related vaccine antigen Pfs25. The lack of structural definition of Pfs28 and the immune response to this candidate has limited further malaria vaccine development for this antigen.

Neutralizing and interfering human antibodies define the structural and mechanistic basis for antigenic diversion.

Defining mechanisms of pathogen immune evasion and neutralization are critical to develop potent vaccines and therapies. Merozoite Surface Protein 1 (MSP-1) is a malaria vaccine antigen and antibodies to MSP-1 are associated with protection from disease. However, MSP-1-based vaccines performed poorly in clinical trials in part due to a limited understanding of the protective antibody response to MSP-1 and of immune evasion by antigenic diversion.

Design of the SARS-CoV-2 RBD vaccine antigen improves neutralizing antibody response.

The receptor binding domain (RBD) of the SARS-CoV-2 spike protein is the primary target of neutralizing antibodies and is a component of almost all current vaccines. Here, RBD immunogens were created with stabilizing amino acid changes that improve the neutralizing antibody response, as well as characteristics for production, storage, and distribution. A computational design and in vitro screening platform identified three improved immunogens, each with approximately nine amino acid changes relative to the native RBD sequence, and four key changes conserved between immunogens.

Design of the SARS-CoV-2 RBD vaccine antigen improves neutralizing antibody response.

The receptor binding domain (RBD) of the SARS-CoV-2 spike protein is the primary target of neutralizing antibodies and is a component of almost all vaccine candidates. Here, RBD immunogens were created with stabilizing amino acid changes that improve the neutralizing antibody response, as well as characteristics for production, storage, and distribution. A computational design and screening platform identified three improved immunogens, each with approximately nine amino acid changes relative to the native RBD sequence and four key changes conserved between immunogens.

A human monoclonal antibody blocks malaria transmission and defines a highly conserved neutralizing epitope on gametes.

Malaria elimination requires tools that interrupt parasite transmission. Here, we characterize B cell receptor responses among Malian adults vaccinated against the first domain of the cysteine-rich 230 kDa gamete surface protein Pfs230, a key protein in sexual stage development of P. falciparum parasites.

Crystal structure of P. falciparum Cpn60 bound to ATP reveals an open dynamic conformation before substrate binding.

Plasmodium falciparum harbors group 1 and group 2 chaperonin systems to mediate the folding of cellular proteins in different cellular locations. Two distinct group 1 chaperonins operate in the organelles of mitochondria and apicoplasts, while group 2 chaperonins function in the cytosol. No structural information has been reported for any chaperonin from plasmodium.

Structures of AAA protein translocase Bcs1 suggest translocation mechanism of a folded protein.

The mitochondrial membrane-bound AAA protein Bcs1 translocate substrates across the mitochondrial inner membrane without previous unfolding. One substrate of Bcs1 is the iron-sulfur protein (ISP), a subunit of the respiratory Complex III. How Bcs1 translocates ISP across the membrane is unknown.

Blood-Stage Malaria Parasite Antigens: Structure, Function, and Vaccine Potential.

Plasmodium parasites are the causative agent of malaria, a disease that kills approximately 450,000 individuals annually, with the majority of deaths occurring in children under the age of 5 years and the development of a malaria vaccine is a global health priority. Plasmodium parasites undergo a complex life cycle requiring numerous diverse protein families. The blood stage of parasite development results in the clinical manifestation of disease.

Structural Basis of p97 Inhibition by the Site-Selective Anticancer Compound CB-5083.

Inhibition of p97, a key player in the ubiquitin-proteasome degradation pathway, has been proposed as a treatment of cancer. This concept was nearly realized recently when a potent p97 inhibitor, 1-[4-(benzylamino)-5H,7H,8H-pyrano[4,3-d]pyrimidin-2-yl]-2-methyl-1H-indole-4-carboxamide (CB-5083), was developed and demonstrated broad antitumor activity in various tumor models. CB-5083 functions as a competitive inhibitor that binds selectively to the ATP-binding site of the D2 domain, although both the D1 and D2 ATPase sites of p97 are highly similar.

Structural basis for nucleotide-modulated p97 association with the ER membrane.

Association of the cytosolic AAA (ATPases associated with various cellular activities) protein p97 to membranes is essential for various cellular processes including endoplasmic reticulum (ER)-associated degradation. The p97 consists of two ATPase domains and an N domain that interacts with numerous cofactors. The N domain of p97 is known to undergo a large nucleotide-dependent conformation switch, but its physiological relevance is unclear.

A Mighty "Protein Extractor" of the Cell: Structure and Function of the p97/CDC48 ATPase.

p97/VCP (known as Cdc48 in or TER94 in ) is one of the most abundant cytosolic ATPases. It is highly conserved from archaebacteria to eukaryotes. In conjunction with a large number of cofactors and adaptors, it couples ATP hydrolysis to segregation of polypeptides from immobile cellular structures such as protein assemblies, membranes, ribosome, and chromatin.

Mutations in the Human AAA Chaperone p97 and Related Diseases.

A number of neurodegenerative diseases have been linked to mutations in the human protein p97, an abundant cytosolic AAA (ATPase associated with various cellular activities) ATPase, that functions in a large number of cellular pathways. With the assistance of a variety of cofactors and adaptor proteins, p97 couples the energy of ATP hydrolysis to conformational changes that are necessary for its function. Disease-linked mutations, which are found at the interface between two main domains of p97, have been shown to alter the function of the protein, although the pathogenic mutations do not appear to alter the structure of individual subunit of p97 or the formation of the hexameric biological unit.

Structures of the Multidrug Transporter P-glycoprotein Reveal Asymmetric ATP Binding and the Mechanism of Polyspecificity.

P-glycoprotein (P-gp) is a polyspecific ATP-dependent transporter linked to multidrug resistance in cancer; it plays important roles in determining the pharmacokinetics of many drugs. Understanding the structural basis of P-gp, substrate polyspecificity has been hampered by its intrinsic flexibility, which is facilitated by a 75-residue linker that connects the two halves of P-gp. Here we constructed a mutant murine P-gp with a shortened linker to facilitate structural determination.

Hydrogen Bonding to the Substrate Is Not Required for Rieske Iron-Sulfur Protein Docking to the Quinol Oxidation Site of Complex III.

Complex III or the cytochrome (cyt) bc complex constitutes an integral part of the respiratory chain of most aerobic organisms and of the photosynthetic apparatus of anoxygenic purple bacteria. The function of cyt bc is to couple the reaction of electron transfer from ubiquinol to cytochrome c to proton pumping across the membrane. Mechanistically, the electron transfer reaction requires docking of its Rieske iron-sulfur protein (ISP) subunit to the quinol oxidation site (Q) of the complex.

Structure and function of the AAA+ ATPase p97/Cdc48p.

p97 (also known as valosin-containing protein (VCP) in mammals or Cdc48p in Saccharomyces cerevisiae) is an evolutionarily conserved ATPase present in all eukaryotes and archaebacteria. In conjunction with a collection of cofactors and adaptors, p97/Cdc48p performs an array of biological functions mostly through modulating the stability of 'client' proteins. Using energy from ATP hydrolysis, p97/Cdc48p segregates these molecules from immobile cellular structures such as protein assemblies, membrane organelles, and chromatin.

Role of the D1-D2 Linker of Human VCP/p97 in the Asymmetry and ATPase Activity of the D1-domain.

Human AAA(+) protein p97 consists of an N-domain and two tandem ATPase domains D1 and D2, which are connected by the N-D1 and the D1-D2 linkers. Inclusion of the D1-D2 linker, a 22-amino acid peptide, at the end of p97 N-D1 truncate has been shown to activate ATP hydrolysis of its D1-domain, although the mechanism of activation remains unclear. Here, we identify the N-terminal half of this linker, highly conserved from human to fungi, is essential for the ATPase activation.

Molecular Basis for Cooperative Binding of Anionic Phospholipids to the PH Domain of the Arf GAP ASAP1.

We have defined the molecular basis for association of the PH domain of the Arf GAP ASAP1 with phospholipid bilayers. Structures of the unliganded and dibutyryl PtdIns(4,5)P2-bound PH domain were solved. PtdIns(4,5)P2 made contact with both a canonical site (C site) and an atypical site (A site).

Altered intersubunit communication is the molecular basis for functional defects of pathogenic p97 mutants.

The human AAA ATPase p97 is a molecular chaperone essential in cellular proteostasis. Single amino acid substitutions in p97 have been linked to a clinical multiple-disorder condition known as inclusion body myopathy associated with Paget's disease of the bone and frontotemporal dementia. How the mutations affect the molecular mechanism that governs the function of p97 remains unclear.