Advances in Fiber Diffraction and General Methods

Conference: 2020: 70th ACA Annual Meeting
08/02/2020: 12:00 PM  - 3:00 PM 
1.2.4 
Oral Session 
Virtual  

Presentations

Opening Remarks

12:00 PM - 12:03 PM 

Advanced Methodology and Measurements of Material and Mechanical Properties of Heart Valves Under Dynamic Strain

12:03 PM - 12:15 PM 
Mammalian heart valves are complex soft tissue assemblies with diffuse boundaries between constituent tissue types, i.e. leaflet (LL), chordae tendinae (CT) and papillary muscles (PM). In mitral and tricuspid valves, the LL are connected to the CT which connect to the ventricular wall on the other end by interfacing with PM. Each of these constituent tissue elements are markedly different in molecular packing and architecture, and hence mechanical properties from these tissue boundaries or interfaces. The material composition and the mechanical properties, as reported here, were not previously available for these tissue assemblies. We present here, developments in multi-scale methodologies using X-diffraction mapping and microscopic evaluation of the application of dynamic strain on these assemblies. The correlation between changes in "molecular strain" in comparison to macro and microscopic strain provide insights in to possible points of failure in these tissues. This information is of great value in developing novel heart valve implants, improving the surgical simulators and aide in developing new methods of teaching surgical techniques, and assisting with robot-assisted surgery. 

View Proposal 129

Author

Rama Sashank Madhurapantula, Illinois Institute of Technology Chicago, IL 

Additional Author

Joseph Orgel Chicago, IL 

Cryo-EM and protein-protein docking reveal myosin loop 4 contacts actin and tropomyosin on thin filaments

12:15 PM - 12:30 PM 
Cardiac muscle contraction occurs when thin filaments slide past thick filaments driven by the action of the molecular motor, myosin. Projecting from thick filaments, myosin heads bind to thin filament actin and undergo ATPase-associated conformational changes to propel the filaments to move past one another. Tropomyosin, a coiled-coil protein that wraps around actin as a continuous cable, regulates this process by sterically controlling myosin's ability to bind to actin. Although several actomyosin structures have been proposed, the structure describing cardiac actin – myosin interactions including tropomyosin has not been solved. Here, we present the structure of the cardiac actomyosin-tropomyosin complex in the nucleotide-free state, solved to a resolution of 4.2 Å using cryo-electron microscopy and helical reconstruction. The atomic model derived from this reconstruction shows a strong, mostly hydrophobic interface between actin and myosin that is consistent with previously determined non-cardiac structures. However, we were able to obtain additional residue-residue discrimination, by combining our cryo-EM structure with results of protein-protein docking methods, revealing novel electrostatic interactions between myosin loop 4 (363-376) and tropomyosin. This hybrid method shows that the tip of myosin loop 4, most notably residue Arg369, forms distinct interactions between successive tropomyosin segments on actin protomers. We propose that these favorable contacts compete with and displace ones normally found between actin and tropomyosin in the absence of myosin, playing a vital role in the transition from the relaxed to contracted states. The impact of these structural studies is twofold: (1) they suggest that the favorable myosin loop 4 – tropomyosin interaction restructures the thin filament during the actin-myosin ATPase cycle; (2) they lay a foundation for understanding molecular mechanisms by which disease-rendering point mutations perturb normal actin-myosin-tropomyosin binding. 

View Proposal 290

Author

Matthew Doran

Additional Author(s)

William Lehman, Boston University Boston, MA 
Esther Bullitt, Boston University Boston, MA 
Michael Rynkiewicz, Boston University Boston, MA 
Elumalai Pavadai, Boston University Boston, MA 
Michael Regnier, University of Washington Seattle, WA 
Michael Geeves, University of Kent
Jeff Moore, University of Massachusetts Lowell
Jonny Walklate, University of Kent

X-ray fiber diffraction as a tool to study Nemaline Myopathy, a debilitating muscle disease

12:30 PM - 1:00 PM 
Nemaline myopathy (NM) is one of the most common congenital non-dystrophic human muscle diseases and is characterized by severe muscle weakness and the presence of nemaline bodies (rods) in skeletal muscle biopsies. In this talk I will highlight two recent X-ray diffraction studies done at the BioCAT Beamline 18ID at the Advanced Photon Source ANL that have yielded new insights into the pathogenic mechanisms in these debilitating diseases. Both of these studies showed that the muscle dysfunction has its basis in changes in the structure of the actin containing thin filaments. One form of nemaline myopathy is caused by mutations in the KBTBD13 (NEM6) gene. The role of KBTBD13 in muscle is unknown. An international team of investigators recently reported In de Winter J et al. 2020 ( J Clin Invest. 130(2):754-767 how a combination of transcranial magnetic stimulation-induced muscle relaxation, muscle fiber- and sarcomere-contractility assays, super-resolution microscopy, and low angle X-ray diffraction revealed that the impaired muscle relaxation kinetics in NEM6 patients are caused by structural changes in the thin filament that are the fundamental cause of muscle weakness. Other forms of NM are due to mutations in nebulin , a giant protein that winds around the actin filaments in the sarcomeres of skeletal muscle. The authors of a second study in Nature Communications (Lindquist et al., Nat Commun. 2020 Jun 1;11(1):2699.) created a mouse model that mimics the typical nebulin-based NM patient with compound-heterozygous mutations. Functional, structural, and biochemical studies revealed altered thin filament structure, increased myofilament lattice spacing, a reduced myofibrillar fractional area, and reduced force production. In particular, X-ray diffraction studies revealed that the actin filament is twisted with a larger radius, that tropomyosin and troponin behavior is altered, and that the myofilament spacing is increased, again showing that the muscle weakness in nemaline myopathy is caused by changes in thin filament structure. These results will be discussed in the context of technical advances at that BioCAT that enabled these studies along with some future directions. 

View Proposal 262

Author

Thomas Irving, Biology, Illinois Inst of Technology Chicago, IL 

Coffee Break

1:00 PM - 1:30 PM 

Macromolecular X-ray crystallography: soon to be a road less traveled?

1:30 PM - 1:45 PM 
As a young(ish) structural biologist, I have increasingly become reflective about the longevity of macromolecular crystallography and even whether, perhaps, I might hear its swan song, rather than crystallography hearing mine. Here, I will present my current thoughts about this question and also give some insight into the use of the Protein Data Bank as a data source. The results do suggest that a change in the landscape of structural biology appears to be in progress with the rise of cryo-electron microscopy outcompeting crystallography for new labor and talent. 

View Proposal 263

Author

John Beale

X-ray diffraction reveals the mechanical load tolerance of mammalian neuronal tissues in traumatically induced injury

1:45 PM - 2:15 PM 
Those living with traumatically induced injuries, including but not limited to, Traumatic Brain Injury (TBI) face an elevated risk for developing chronic health issues including Alzheimer's disease (AD) or AD-like dementia and depression. In an attempt to help address these concerns, we have employed an X-ray diffraction (XRD) scanning methodology to detect the mechanical load threshold/s at which meaningful structural changes occur to mammalian neuronal tissues. Systematically loaded animal models of intact brain and extracted tissues (both neuronal and connective) were then sampled with XRD and conventional microscopy to help confirm and validate the methodologies and the initial observations. The observations made so far provide a means to begin the process of integrating primary mechanical damage criteria into the current and ongoing computational efforts to better understand the outcomes to traumatic injuries. 

View Proposal 127

Author

Joseph Orgel Chicago, IL 

Exploring mechanisms of insulin-degrading enzyme activation and localization in the degradation of amyloid beta and insulin.

2:15 PM - 2:30 PM 
The amyloid beta peptide is central to the etiology of Alzheimer's disease, and the small number of proteolytic enzymes that degrade it are therefore of considerable interest. Insulin-degrading enzyme (IDE) is a zinc metallopeptidase that, in additional to metabolizing amyloid beta, is responsible for degrading insulin and likely other bioactive peptides. Efforts are underway to target IDE for the treatment of Alzheimer's disease and diabetes, primarily by developing substrate specific activators or inhibitors. We have characterized IDE activation by both small peptides and anions, particularly polyanions, and obtained crystal structures that define both allosteric binding sites. In addition, a key question is how IDE produced in the cytosol gains access to amyloid beta and insulin, which are taken up by the cell into endocytic compartments. We present recent evidence that IDE may partially localize to endosomes by binding the head groups of phosphatidylinositol phosphates located on the organelle outer membranes. Crystal structures with bound phosphatidylinositol phosphate and inositol phosphate illuminate aspects of the localization mechanism as well as activation by anions. 

View Proposal 188

Author

David Rodgers, University of Kentucky Lexington, KY 

Additional Author(s)

Louis Hersh, University of Kentucky Lexington, KY 
Emilia Galperin, University of Kentucky Lexington, KY 
Eun Suk Song, University of Kentucky Lexington, KY 

High resolution cryo-EM structure of the EspA filament from EPEC: revealing the mechanism of effector translocation in the type 3 secretion system

2:30 PM - 2:45 PM 
Contributors: Bronwyn Lyons, Claire Atkinson, Wanyin Deng, Antonio Serapio Palacios, Brett Finlay, Natalie C.J. Strynadka Antimicrobial resistance (AMR) is a growing concern for the global population, predominantly in animal husbandry, hospitals, and developing countries. Several pathogens are of particular concern, including Pseudomonas aeruginosa and Enterobacter spp. (Escherichia coli). The rise in AMR has contributed to the decline in development of novel antibiotics, thus pushing the world closer to a pre-antibiotic era. Virulence mechanisms that bacteria use are often not required for their survival and therefore are promising targets for the development of anti-virulence compounds. The type III secretion system (T3SS) is a highly conserved virulence mechanism employed by several Gram-negative pathogens, such as those discussed above. The T3SS is a syringe-like proteinaceous channel that spans the inner and outer membranes of the bacterial cell, projecting into the extracellular medium where it interacts with the host cell membrane to deliver proteinaceous virulence factors. The main components of the T3SS are: (1) the basal body, spanning the inner and outer membranes of the bacterial cell; (2) the needle that projects from the basal body and secretin lumen, and (3) the tip and pore-forming translocon that completes the continuous channel from bacteria to host. Entero-pathogenic and -hemorrhagic E. coli (EPEC/EHEC) possess a distinct T3SS from that of other species; EPEC/EHEC possess a needle extension, or filament, in place of the 'tip' component. This filament, called EspA, can be upwards of 700 nm in length, extending the channel from the needle through EspA and connecting to the translocon upon host-cell contact. It is thought that the EspA filament may provide a more flexible extension of the conduit to protect effectors in the unfavourable environment of the gut epithelium. The function of EspA is absolutely required for colonization of EPEC within the gut epithelium and espA EPEC knockouts are associated with decreased virulence. Here we describe the cryo-EM structure of natively-sheared EspA filaments from EPEC, determined to 3.6 Å resolution. Data were collected at the High-Resolution Macromolecular Electron Microscopy (HRMEM) facility at the University of British Columbia. The EspA protomer is described as a coiled-coil with each helix connected by an insertion domain that decorates the exterior of the filament. Within the filament lumen, a pattern of positively charged residues adjacent to a hydrophobic groove lines the lumen of the filament in a spiral manner, suggesting a mechanism of translocation through the channel mediated via electrostatics. Using structure-guided mutagenesis of these residues, in vivo studies corroborate the role of these residues in secretion function. Interestingly, the EspA filament is distinct from the needle structure, in that it possesses a helix-turn-strand insertion that connects the two helices of the conserved coiled-coil rather than a short loop as seen in the recent Salmonella PrgI needle structure, or a globular domain as seen in other non-filamentous orthologs. The high-resolution structure of the EspA filament will aid in structure-guided drug design of novel antivirulence therapeutics. This may lead to improved prophylactic treatment of EHEC/EPEC infections, which are on the rise due to increased produce contamination. 

View Proposal 287

Author

Bronwyn Lyons, University of British Columbia Vancouver, BC 

Patient derived Fab structures suggest mechanism by which affinity maturation promotes autoantibody recognition of MuSK in the autoimmune disease myasthenia gravis

2:45 PM - 3:00 PM 
Myasthenia gravis (MG) is a chronic autoimmune disorder affecting neuromuscular transmission caused by pathogenic autoantibodies targeting components of the neuromuscular junction. Pathogenic autoantibodies against the muscle-specific tyrosine kinase (MuSK) are predominantly of the IgG4 subclass, which have the unique ability to participate in Fab-arm exchange making them functionally monovalent. Recent isolation of human MuSK monoclonal autoantibodies (mAbs) from patients with MG has allowed for the characterization of MuSK-specific autoantibodies. Using X-ray crystallography we have structurally characterized two patient derived Fabs from MG. Our data suggests that affinity maturation increases the negative charge of the CDR loops which increases Fab affinity to the positively charged second Ig-like domain in MuSK. Affinity maturation leads to sub-nanomolar binding to the autoantigen, while unmutated common ancestors (UCA) Fabs have an approximately 100 fold lower affinity than their respective mature fabs. In acetylcholine receptor clustering assays mature mAbs, UCA mAbs, and mature Fabs bound the autoantigen with pathogenic capacity, while monovalent UCA Fabs bound the autoantigen without measurable pathogenic capacity. These findings suggest that high affinity, functionally monovalent, Fab-arm exchanged IgG4 antibodies tightly bind to the autoantigen MuSK in MuSK MG, thereby preventing MuSK dimerization and downstream signaling, rather than receptor cross linking and activation. 

View Proposal 298

Author

Casey Vieni, New York University School of Medicine New York, NY 

Additional Author(s)

Miriam Fichtner, Department of Immunobiology, Yale University School of Medicine New Haven, CT 
Rachel Redler, New York University Grossman School of Medicine New York, NY 
Ljuvica Kolich, New York University Grossman School of Medicine New York, NY 
Steven Burden, New York University Grossman School of Medicine New York, NY 
Kevin O'Connor, Department of Immunobiology, Yale University School of Medicine New Haven, CT 
Damian Ekiert