Hot Structures

Conference: 2020: 70th ACA Annual Meeting
08/07/2020: 12:00 PM  - 3:00 PM 
3.2.3 
Oral Session 
Virtual  

Description

The Hot Structures session will feature talks selected from submitted abstracts describing the newest results from structural studies of biologically important macromolecules. Submissions are welcome that describe high-impact structures which provide insights into structure-function relationships, new biological insights and mechanisms, and methods development. Studies may include the use of X-ray crystallography, XFEL, hybrid methods, and cryo-electron microscopy.

Presentations

Structure of the RECK CC domain, an evolutionary anomaly

12:00 PM - 12:20 PM 
Five small and homologous protein domains (the CC-domains) at the N-terminus of the RECK (Reversion-inducing Cysteine-rich Protein with Kazal Motifs), a multi-domain glycosyl-phosphatidyl-inositol (GPI)-anchored protein, play essential roles in signaling by WNT7A and WNT7B in the context of central nervous system angiogenesis and blood-brain barrier formation and maintenance (1-3). Here, we have developed an integrated strategy to determine the crystal structure of CC domain 4 (CC4) and find that it folds into a compact 4-helix bundle with three disulfide bonds (4). The CC4 structure, together with homology modeling of CC1, reveals the surface locations of critical residues that were shown in previous mutagenesis studies (5-8) to mediate G-protein coupled receptor (GPR)124 binding and WNT7A/WNT7B recognition and signaling. Surprisingly, sequence and structural homology searches reveal no other cell-surface or secreted domains in vertebrates that resemble the CC domain, a pattern that is in striking contrast to other ancient and similarly sized domains, such as Epidermal Growth Factor, Fibronectin Type 3, Immunoglobulin, and Thrombospondin type 1 domains, which are collectively present in hundreds of proteins. References: 1. J. Oh et al., The membrane-anchored MMP inhibitor RECK is a key regulator of extracellular matrix integrity and angiogenesis. Cell 107, 789-800 (2001). 2. T. Muraguchi et al., RECK modulates Notch signaling during cortical neurogenesis by regulating ADAM10 activity. Nat Neurosci 10, 838-845 (2007). 3. B. Vanhollebeke et al., Tip cell-specific requirement for an atypical Gpr124- and Reck-dependent Wnt/beta-catenin pathway during brain angiogenesis. Elife 4, (2015). 4. TH. Chang, FL. Hsieh, P. M. Smallwood, S. G. Gabelli, J. Nathans, Structure of the RECK CC domain, an evolutionary anomaly. Proc Natl Acad Sci U S A, (2020). In press 5. C. Cho, P. M. Smallwood, J. Nathans, Reck and Gpr124 Are Essential Receptor Cofactors for Wnt7a/Wnt7b-Specific Signaling in Mammalian CNS Angiogenesis and Blood-Brain Barrier Regulation. Neuron 95, 1056-1073 e1055 (2017). 6. M. Eubelen et al., A molecular mechanism for Wnt ligand-specific signaling. Science 361, (2018). 7. M. Vallon et al., A RECK-WNT7 Receptor-Ligand Interaction Enables Isoform-Specific Regulation of Wnt Bioavailability. Cell Rep 25, 339-349 e339 (2018). 8. C. Cho, Y. Wang, P. M. Smallwood, J. Williams, J. Nathans, Molecular determinants in Frizzled, Reck, and Wnt7a for ligand-specific signaling in neurovascular development. Elife 8, (2019). 

View Proposal 214

Author

Tao-Hsin Chang

Additional Author(s)

Fu-Lein Hsieh, Johns Hopkins University School of Medicine
Philip Smallwood, Johns Hopkins University School of Medicine
Sandra Gabelli, Johns Hopkins University Ellicott City, MD 
Jeremy Nathans, Johns Hopkins University School of Medicine

Targeting Fe-S Protein to Fight Neglected Tropical Diseases

12:20 PM - 12:40 PM 
Here we describe a novel [4Fe-4S] cluster-containing metalloprotein: cytosolic fumarate hydratase from Leishmania major (LmFH-2). Like aconitase, it uses a [4Fe-4S] cluster to coordinate substrate but is not structurally similar to aconitase. In fact, LmFH-2 has a unique protein fold. Importantly, metal-dependent FHs are found in parasites that cause Neglected Tropical Diseases (NTD), such as leishmaniases, Chagas disease, and sleeping sickness, whereas humans employ a metal-independent FH. NTDs affect approximately one-sixth of the world's population, and due to the appearance of resistance to current medicines, there is an increased interest in the development of innovative strategies to combat NTD. To explore the potential of FHs as anti-NTD targets, we have determined a series of structures of Leishmania major FH, including a structure with the substrate malate bound to the unique iron of the cluster. Our structures provide insights into the catalytic mechanism of class I (metal-dependent) FHs, a mechanism that is likely to be conserved among FHs from NTD-causing organisms. 

View Proposal 289

Author

Patricia Feliciano, MIT/HHMI Cambridge, MA 

Additional Author

Catherine Drennan, MIT

A Catalog of Human Gut Microbial Sulfatases Reveals Structural Features Essential for Endobiotic Processing

12:40 PM - 1:00 PM 
Human gut microbiome-encoded sulfatases are poised to reverse the action of mammalian sulfotransferases that inactivate endo- and xenobiotics as part of Phase II drug metabolism, impacting health and disease. In spite of their health implications, the numbers, types, and diversity of sulfatases within the human gut microbiome remain undefined. Here we present the first catalogue of sulfatase enzymes from the fecal metagenomic sequencing data within the Human Microbiome Project (HMP). Using protein structure-guided methods, we identified 728 distinct microbiome-encoded sulfatase proteins from the 4.8 million unique proteins present in the HMP Stool Sample database. We cloned, expressed, and purified a representative set of these microbial sulfatases and determined two novel crystal structures to pinpoint unique structural motifs essential for the processing of endobiotic-sulfate substrates. We show that gut microbial sulfatases differentially process hormone-sulfates in a manner dependent on active site architecture and quaternary structure. Finally, we demonstrate the inhibitors of human sulfatases have no effect on the microbial enzymes examined here. Taken together, data presented here provides unprecedented insights into the structural and functional diversity of sulfatases encoded by the human gut microbiome. 

View Proposal 113

Author

Samantha Ervin, University of North Carolina at Chapel Hill Carrboro, NC 

Coffee Break

1:00 PM - 1:20 PM 

RNA Duplex Recognition and Unwinding by DEAD-Box Helicase DDX3X

1:20 PM - 1:50 PM 
DEAD-box helicases (DDXs) regulate RNA processing and metabolism by unwinding short double-stranded (ds) RNAs. Sharing a helicase core composed of two RecA-like domains (D1D2), DDXs function in an ATP-dependent, non-processive manner. As an attractive target for cancer and AIDS treatment, DDX3X and its orthologs are extensively studied, yielding a wealth of biochemical and biophysical data, including structures of apo-D1D2 and post-unwound D1D2:single-stranded RNA complex, and the structure of a D2:dsRNA complex that is thought to represent a pre-unwound state. However, the structure of a pre-unwound D1D2:dsRNA complex remains elusive, and thus, the mechanism of DDX action is not fully understood. Here, we describe the structure of a D1D2 core in complex with a 23-base pair dsRNA at pre-unwound state, revealing that two DDXs recognize a 2-turn dsRNA, each DDX mainly recognizes a single RNA strand, and conformational changes induced by ATP binding unwinds the RNA duplex in a cooperative manner. Human DDX3X and its yeast ortholog Ded1p represent a subfamily of DDXs. The worldwide effort in structural and mechanistic analysis of these two DDXs demonstrated that they function as a trimer wherein the protomers play distinct loading and unwinding roles, but the exact number of protomers involved in each role is not clear. Based on new structural and functional data, we conclude that one protomer is responsible for loading and the other two are responsible for unwinding the RNA duplex. 

View Proposal 216

Author

Xinhua Ji, National Cancer Institute, NIH Frederick, MD 

Additional Author(s)

He Song, National Cancer Institute, NIH Frederick, MD 
Lan Jin, National Cancer Institute, NIH Frederick, MD 
Anjana Ram, National Cancer Institute, NIH Frederick, MD 

Bacterial pseudokinase catalyzes protein polyglutamylation to inhibit the SidE-family ubiquitin ligases

1:50 PM - 2:20 PM 
Enzymes with a protein kinase fold transfer phosphate from adenosine 5′-triphosphate (ATP) to substrates in a process known as phosphorylation. Here, we show that the Legionella meta-effector SidJ adopts a protein kinase fold, yet unexpectedly catalyzes protein polyglutamylation. SidJ is activated by host-cell calmodulin to polyglutamylate the SidE family of ubiquitin (Ub) ligases. Crystal structures of the SidJ-calmodulin complex reveal a protein kinase fold that catalyzes ATP-dependent isopeptide bond formation between the amino group of free glutamate and the γ-carboxyl group of an active-site glutamate in SidE. We show that SidJ polyglutamylation of SidE, and the consequent inactivation of Ub ligase activity, is required for successful Legionella replication in a viable eukaryotic host cell. 

View Proposal 314

Author

Diana Tomchick, UT Southwestern Medical Center Irving, TX 

Additional Author(s)

Miles H. Black, UT Southwestern Medical Center Dallas, TX 
Adam Osinski, UT Southwestern Medical Center Dallas, TX 
Marcin Gradowski, Warsaw University of Life Sciences Warsaw
Kelly A. Servage, UT Southwestern Medical Center Dallas, TX 
Krzysztof Pawlowski, Warsaw University of Life Sciences Warsaw
Vincent S. Tagliabracci, UT Southwestern Medical Center Dallas, TX 

HIV-1 CD4 binding site antibody N49P6 mimics CD4 in its quaternary interactions with the HIV-1 Envelope trimer.

2:20 PM - 2:40 PM 
The first step in the HIV-1 entry process is the attachment of the Envelope (Env) trimer to target cell CD4. As such, the CD4 binding site (CD4bs) remains one of the only universally accessible sites on the Env trimer. Few antibodies (Abs) are able to capitalize on this however, due the steric constraints involved in accessing the CD4bs. We recently characterized a near pan neutralizing Ab isolated from the plasma of a HIV-1 "elite neutralizer", N49P7. N49P7 combines many characteristic CD4bs Ab features along with unique interactions to the highly conserved gp120 inner domain Layer 3 to achieve its remarkable neutralization breath and potency. Here we characterize the Ab N49P6 from the same donor and show that N49P6 utilizes many of these characteristics to achieve similar breath. Further, we determined the structure of N49P6 in complex with BG505 SOSIP and show that N49P6 mimics CD4 in its initial quaternary contacts with the neighboring gp120 in the trimer. The details of these interactions pave the way to the creation of the next generation of HIV-1 neutralizing Abs for the use in preformed vaccines and HIV-1 therapeutics. 

View Proposal 252

Author

William Tolbert

Additional Author(s)

Dung Nguyen, Uniformed Services University of the Health Sciences Bethesda, MD 
Zahra Tehrani, Institute of Human Virology, University of Maryland School of Medicine Baltimore, MD 
Mohammad Sajadi, Institute of Human Virology, University of Maryland School of Medicine and Department of Medicine, B Baltimore, MD 
Marzena Pazgier, Uniformed Services University of the Health Sciences Bethesda, MD 

Structure of MlaFEDB lipid transporter reveals an ABC exporter fold and two bound phospholipids

2:40 PM - 3:00 PM 
In double-membraned bacteria, phospholipids must be transported across the cell envelope to maintain the outer membrane barrier, which plays a key role in antibiotic resistance and pathogen virulence. The Mla pathway has been implicated in phospholipid trafficking and outer membrane integrity, but the mechanism by which this ABC transporter facilitates phospholipid transport remains unknown. Using a combination of X-ray crystallography and cryo-EM, we have determined the structure of MlaFEDB inner membrane complex. Our structures reveal that the core transporter subunit, MlaE, adopts an ABC exporter fold and is related to the lipopolysaccharide export system as well as the human lipid exporter families, ABCA and ABCG. Unexpectedly, two phospholipids are bound in the outward-facing pocket of MlaFEDB, raising the possibility that multiple lipid substrates may be translocated each transport cycle. The unusual configuration of bound lipids suggests that lipid reorientation may occur before extrusion through the hydrophobic MlaD pore. Site-specific crosslinking confirms that lipids bind in this pocket in vivo. Structures of the ATPase and regulatory subunits suggest novel mechanisms for the regulation of ABC transporter activity. Our work supports a model in which the Mla system may drive phospholipid export to the bacterial outer membrane, and provides insight into the function of an exporter family conserved from bacteria to humans. 

View Proposal 265

Author

Damian Ekiert

Additional Author(s)

Nicolas Coudray, NYU School of Medicine New York, NY 
Georgia Isom, NYU School of Medicine New York, NY 
Mark MacRae, NYU School of Medicine New York, NY 
Mariyah Saiduddin, NYU School of Medicine New York, NY 
Gira Bhabha, NYU School of Medicine New York, NY