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Monovalent IgG4 autoantibodies require self-antigen driven affinity maturation to acquire pathogenic capacity [Meeting Abstract]
Fichtner, M F L; Vieni, C; Redler, R L; Jiang, R; Suarez, P; Nowak, R; Burden, S J; Bhabha, G; Ekiert, D C; O'Connor, K C
INTRODUCTION The mechanisms underlying B cell-mediated autoimmune disease and the origin of autoreactive B cells are not well understood. Human monoclonal autoantibodies (mAbs) are valuable tools for investigating both. In this study, we used mAbs derived from patients with the autoimmune disorder, myasthenia gravis (MG). A subset of patients with MG have pathogenic autoantibodies that recognize muscle specific tyrosine kinase (MuSK). The autoantibodies of MuSK MG are predominantly of the IgG4 subclass and functionally monovalent as a result of Fab-arm exchange. OBJECTIVE To gain insight into the origin and development of these unique autoantibodies. METHODS AND RESULTS We examined MG patient-derived mAbs, their corresponding germline-encoded unmutated common ancestors (UCA) and monovalent antigen-binding fragments (Fabs) to investigate how antigen-driven affinity maturation contributes to both binding and immunopathology. Mature mAbs, their UCA counterparts and mature monovalent Fabs bound to the MuSK autoantigen and retained their pathogenic capacity. However, monovalent UCA Fabs, which still bound the autoantigen, lost their pathogenic capacity. The mature Fabs were characterized by very high affinity (sub-nanomolar) driven by a rapid on-rate and slow off-rate. However, the UCA affinity was approximately 100-fold less than the mature Fabs, which was driven by a rapid off-rate. SUMMARY/CONCLUSION These findings indicate that the autoantigen initiates the autoimmune response in MuSK MG and drives autoimmunity through the accumulation of somatic hypermutations such that monovalent IgG4 Fab-arm exchanged MG autoantibodies reach a high affinity threshold required for pathogenic capacity
EMBASE:633107896
ISSN: 1550-6606
CID: 4638832
LetB Structure Reveals a Tunnel for Lipid Transport across the Bacterial Envelope
Isom, Georgia L; Coudray, Nicolas; MacRae, Mark R; McManus, Collin T; Ekiert, Damian C; Bhabha, Gira
Gram-negative bacteria are surrounded by an outer membrane composed of phospholipids and lipopolysaccharide, which acts as a barrier and contributes to antibiotic resistance. The systems that mediate phospholipid trafficking across the periplasm, such as MCE (Mammalian Cell Entry) transporters, have not been well characterized. Our ~3.5 Å cryo-EM structure of the E. coli MCE protein LetB reveals an ~0.6 megadalton complex that consists of seven stacked rings, with a central hydrophobic tunnel sufficiently long to span the periplasm. Lipids bind inside the tunnel, suggesting that it functions as a pathway for lipid transport. Cryo-EM structures in the open and closed states reveal a dynamic tunnel lining, with implications for gating or substrate translocation. Our results support a model in which LetB establishes a physical link between the two membranes and creates a hydrophobic pathway for the translocation of lipids across the periplasm.
PMID: 32359438
ISSN: 1097-4172
CID: 4415712
Architectures of Lipid Transport Systems for the Bacterial Outer Membrane
Ekiert, Damian C; Bhabha, Gira; Isom, Georgia L; Greenan, Garrett; Ovchinnikov, Sergey; Henderson, Ian R; Cox, Jeffery S; Vale, Ronald D
How phospholipids are trafficked between the bacterial inner and outer membranes through the hydrophilic space of the periplasm is not known. We report that members of the mammalian cell entry (MCE) protein family form hexameric assemblies with a central channel capable of mediating lipid transport. The E. coli MCE protein, MlaD, forms a ring associated with an ABC transporter complex in the inner membrane. A soluble lipid-binding protein, MlaC, ferries lipids between MlaD and an outer membrane protein complex. In contrast, EM structures of two other E. coli MCE proteins show that YebT forms an elongated tube consisting of seven stacked MCE rings, and PqiB adopts a syringe-like architecture. Both YebT and PqiB create channels of sufficient length to span the periplasmic space. This work reveals diverse architectures of highly conserved protein-based channels implicated in the transport of lipids between the membranes of bacteria and some eukaryotic organelles.
PMCID:5467742
PMID: 28388411
ISSN: 1097-4172
CID: 2530792
Exploring the repeat protein universe through computational protein design
Brunette, T J; Parmeggiani, Fabio; Huang, Po-Ssu; Bhabha, Gira; Ekiert, Damian C; Tsutakawa, Susan E; Hura, Greg L; Tainer, John A; Baker, David
A central question in protein evolution is the extent to which naturally occurring proteins sample the space of folded structures accessible to the polypeptide chain. Repeat proteins composed of multiple tandem copies of a modular structure unit are widespread in nature and have critical roles in molecular recognition, signalling, and other essential biological processes. Naturally occurring repeat proteins have been re-engineered for molecular recognition and modular scaffolding applications. Here we use computational protein design to investigate the space of folded structures that can be generated by tandem repeating a simple helix-loop-helix-loop structural motif. Eighty-three designs with sequences unrelated to known repeat proteins were experimentally characterized. Of these, 53 are monomeric and stable at 95 degrees C, and 43 have solution X-ray scattering spectra consistent with the design models. Crystal structures of 15 designs spanning a broad range of curvatures are in close agreement with the design models with root mean square deviations ranging from 0.7 to 2.5 A. Our results show that existing repeat proteins occupy only a small fraction of the possible repeat protein sequence and structure space and that it is possible to design novel repeat proteins with precisely specified geometries, opening up a wide array of new possibilities for biomolecular engineering.
PMCID:4845728
PMID: 26675729
ISSN: 1476-4687
CID: 2291262
Structure of a PE-PPE-EspG complex from Mycobacterium tuberculosis reveals molecular specificity of ESX protein secretion
Ekiert, Damian C; Cox, Jeffery S
Nearly 10% of the coding capacity of the Mycobacterium tuberculosis genome is devoted to two highly expanded and enigmatic protein families called PE and PPE, some of which are important virulence/immunogenicity factors and are secreted during infection via a unique alternative secretory system termed "type VII." How PE-PPE proteins function during infection and how they are translocated to the bacterial surface through the five distinct type VII secretion systems [ESAT-6 secretion system (ESX)] of M. tuberculosis is poorly understood. Here, we report the crystal structure of a PE-PPE heterodimer bound to ESX secretion-associated protein G (EspG), which adopts a novel fold. This PE-PPE-EspG complex, along with structures of two additional EspGs, suggests that EspG acts as an adaptor that recognizes specific PE-PPE protein complexes via extensive interactions with PPE domains, and delivers them to ESX machinery for secretion. Surprisingly, secretion of most PE-PPE proteins in M. tuberculosis is likely mediated by EspG from the ESX-5 system, underscoring the importance of ESX-5 in mycobacterial pathogenesis. Moreover, our results indicate that PE-PPE domains function as cis-acting targeting sequences that are read out by EspGs, revealing the molecular specificity for secretion through distinct ESX pathways.
PMCID:4205667
PMID: 25275011
ISSN: 1091-6490
CID: 2291272
De novo-designed enzymes as small-molecule-regulated fluorescence imaging tags and fluorescent reporters
Liu, Yu; Zhang, Xin; Tan, Yun Lei; Bhabha, Gira; Ekiert, Damian C; Kipnis, Yakov; Bjelic, Sinisa; Baker, David; Kelly, Jeffery W
Enzyme-based tags attached to a protein-of-interest (POI) that react with a small molecule, rendering the conjugate fluorescent, are very useful for studying the POI in living cells. These tags are typically based on endogenous enzymes, so protein engineering is required to ensure that the small-molecule probe does not react with the endogenous enzyme in the cell of interest. Here we demonstrate that de novo-designed enzymes can be used as tags to attach to POIs. The inherent bioorthogonality of the de novo-designed enzyme-small-molecule probe reaction circumvents the need for protein engineering, since these enzyme activities are not present in living organisms. Herein, we transform a family of de novo-designed retroaldolases into variable-molecular-weight tags exhibiting fluorescence imaging, reporter, and electrophoresis applications that are regulated by tailored, reactive small-molecule fluorophores.
PMCID:4183642
PMID: 25209927
ISSN: 1520-5126
CID: 2291282
Small molecule probes to quantify the functional fraction of a specific protein in a cell with minimal folding equilibrium shifts
Liu, Yu; Tan, Yun Lei; Zhang, Xin; Bhabha, Gira; Ekiert, Damian C; Genereux, Joseph C; Cho, Younhee; Kipnis, Yakov; Bjelic, Sinisa; Baker, David; Kelly, Jeffery W
Although much is known about protein folding in buffers, it remains unclear how the cellular protein homeostasis network functions as a system to partition client proteins between folded and functional, soluble and misfolded, and aggregated conformations. Herein, we develop small molecule folding probes that specifically react with the folded and functional fraction of the protein of interest, enabling fluorescence-based quantification of this fraction in cell lysate at a time point of interest. Importantly, these probes minimally perturb a protein's folding equilibria within cells during and after cell lysis, because sufficient cellular chaperone/chaperonin holdase activity is created by rapid ATP depletion during cell lysis. The folding probe strategy and the faithful quantification of a particular protein's functional fraction are exemplified with retroaldolase, a de novo designed enzyme, and transthyretin, a nonenzyme protein. Our findings challenge the often invoked assumption that the soluble fraction of a client protein is fully folded in the cell. Moreover, our results reveal that the partitioning of destabilized retroaldolase and transthyretin mutants between the aforementioned conformational states is strongly influenced by cytosolic proteostasis network perturbations. Overall, our results suggest that applying a chemical folding probe strategy to other client proteins offers opportunities to reveal how the proteostasis network functions as a system to regulate the folding and function of individual client proteins in vivo.
PMCID:3970509
PMID: 24591605
ISSN: 1091-6490
CID: 2291292
A common solution to group 2 influenza virus neutralization
Friesen, Robert H E; Lee, Peter S; Stoop, Esther J M; Hoffman, Ryan M B; Ekiert, Damian C; Bhabha, Gira; Yu, Wenli; Juraszek, Jarek; Koudstaal, Wouter; Jongeneelen, Mandy; Korse, Hans J W M; Ophorst, Carla; Brinkman-van der Linden, Els C M; Throsby, Mark; Kwakkenbos, Mark J; Bakker, Arjen Q; Beaumont, Tim; Spits, Hergen; Kwaks, Ted; Vogels, Ronald; Ward, Andrew B; Goudsmit, Jaap; Wilson, Ian A
The discovery and characterization of broadly neutralizing antibodies (bnAbs) against influenza viruses have raised hopes for the development of monoclonal antibody (mAb)-based immunotherapy and the design of universal influenza vaccines. Only one human bnAb (CR8020) specifically recognizing group 2 influenza A viruses has been previously characterized that binds to a highly conserved epitope at the base of the hemagglutinin (HA) stem and has neutralizing activity against H3, H7, and H10 viruses. Here, we report a second group 2 bnAb, CR8043, which was derived from a different germ-line gene encoding a highly divergent amino acid sequence. CR8043 has in vitro neutralizing activity against H3 and H10 viruses and protects mice against challenge with a lethal dose of H3N2 and H7N7 viruses. The crystal structure and EM reconstructions of the CR8043-H3 HA complex revealed that CR8043 binds to a site similar to the CR8020 epitope but uses an alternative angle of approach and a distinct set of interactions. The identification of another antibody against the group 2 stem epitope suggests that this conserved site of vulnerability has great potential for design of therapeutics and vaccines.
PMCID:3890827
PMID: 24335589
ISSN: 1091-6490
CID: 2291302
Divergent evolution of protein conformational dynamics in dihydrofolate reductase
Bhabha, Gira; Ekiert, Damian C; Jennewein, Madeleine; Zmasek, Christian M; Tuttle, Lisa M; Kroon, Gerard; Dyson, H Jane; Godzik, Adam; Wilson, Ian A; Wright, Peter E
Molecular evolution is driven by mutations, which may affect the fitness of an organism and are then subject to natural selection or genetic drift. Analysis of primary protein sequences and tertiary structures has yielded valuable insights into the evolution of protein function, but little is known about the evolution of functional mechanisms, protein dynamics and conformational plasticity essential for activity. We characterized the atomic-level motions across divergent members of the dihydrofolate reductase (DHFR) family. Despite structural similarity, Escherichia coli and human DHFRs use different dynamic mechanisms to perform the same function, and human DHFR cannot complement DHFR-deficient E. coli cells. Identification of the primary-sequence determinants of flexibility in DHFRs from several species allowed us to propose a likely scenario for the evolution of functionally important DHFR dynamics following a pattern of divergent evolution that is tuned by cellular environment.
PMCID:3823643
PMID: 24077226
ISSN: 1545-9985
CID: 2291312
Reshaping antibody diversity
Wang, Feng; Ekiert, Damian C; Ahmad, Insha; Yu, Wenli; Zhang, Yong; Bazirgan, Omar; Torkamani, Ali; Raudsepp, Terje; Mwangi, Waithaka; Criscitiello, Michael F; Wilson, Ian A; Schultz, Peter G; Smider, Vaughn V
Some species mount a robust antibody response despite having limited genome-encoded combinatorial diversity potential. Cows are unusual in having exceptionally long CDR H3 loops and few V regions, but the mechanism for creating diversity is not understood. Deep sequencing reveals that ultralong CDR H3s contain a remarkable complexity of cysteines, suggesting that disulfide-bonded minidomains may arise during repertoire development. Indeed, crystal structures of two cow antibodies reveal that these CDR H3s form a very unusual architecture composed of a beta strand "stalk" that supports a structurally diverse, disulfide-bonded "knob" domain. Diversity arises from somatic hypermutation of an ultralong DH with a severe codon bias toward mutation to cysteine. These unusual antibodies can be elicited to recognize defined antigens through the knob domain. Thus, the bovine immune system produces an antibody repertoire composed of ultralong CDR H3s that fold into a diversity of minidomains generated through combinations of somatically generated disulfides.
PMCID:4007204
PMID: 23746848
ISSN: 1097-4172
CID: 2291322