Hot Structures 2

Conference: 2020: 70th ACA Annual Meeting
08/02/2020: 12:00 PM  - 3:00 PM 
5.1.2 
Oral Session 
Virtual  

Description

This session will be comprised of talks describing exciting new results in structural biology. The majority of talks will be selected from submitted abstracts.

Presentations

Direct detection of coupled proton and electron transfers in human manganese superoxide dismutase

12:00 PM - 12:20 PM 
Human manganese superoxide dismutase (MnSOD) is a critical oxidoreductase found in the mitochondria matrix. Concerted proton and electron transfers (CPETs) are used by the enzyme to rid the mitochondria of O2•-, a precursor to other reactive oxygen species (ROS) that are harmful in excessive amounts. The mechanism of CPET-utilizing enzymes are difficult to discern due to the inability to directly detect the protonation states of specific residues and solvent molecules involved in catalysis while controlling the redox state of the enzyme. Here, we utilize neutron diffraction of redox-controlled MnSOD crystals to yield all-atom structures of Mn3+SOD and Mn2+SOD revealing five sites of differential protonations. A novel mechanism is introduced from the direct observation of a glutamine amide-imidic acid tautomerization and three locations of low-barrier hydrogen bonds that change with the oxidation state of the metal. Quantum calculations provide insight into electronic modulation and thermodynamic properties of the observed structures. 

View Proposal 333

Author(s)

Jahaun Azadmanesh, Univ. Nebraska Medical Center Omaha, NE 
Nate Test2, Attendee Interactive Marriotsville, MD 

Additional Author(s)

WIlliam Lutz, University of Nebraska Medical Center Omaha, NE 
Leighton Coates, Oak Ridge National Laboratory Oak Ridge, TN 
Kevin Weiss, Oak Ridge National Laboratory Oak Ridge, TN 
Gloria E O Borgstahl, The Eppley Inst For Cancer Res

Structures of MHC-I/Tapasin and MHC-I/TAPBPR Describe the Mechanism of Peptide Loading Antigen Presentation

12:20 PM - 12:40 PM 
MHC molecules of the class I type (MHC-I) bind to endogenous peptides in the endoplasmic reticulum and the peptide-loaded MHC-I molecules are subsequently transported to the cell surface where they serve as indispensable ligands for T cell and NK cell development and function. Peptide loading occurs in the ER within the peptide loading complex (PLC) in which critical aspects of MHC-I stabilization and peptide loading are dependent on the chaperone, tapasin. However, MHC-I molecules can also be loaded independently of the PLC by a tapasin homolog designated TAPBPR. To elucidate mechanistic aspects of PLC-dependent and independent peptide loading we have determined the X-ray crystal structures of MHC-I with tapasin and compare that with previously reported the structure of a MHC-I/TAPBPR complex (Jiang, et al., Science 2017, 358, 1064-68). These structures capture distinct chaperone-stabilized MHC-I conformation, and provide insight into the mechanism of PLC-dependent and PLC-independent MHC-I peptide loading. (Supported by the Intramural research program of the NIAID, NIH) 

View Proposal 195

Author

Jiansheng Jiang, NIAID/NIH Bethesda, MD 

Additional Author(s)

Kannan Natarajan, LISB/NIAID/NIH Bethesda, MD 
Ellen Kim, LISB/NIAID/NIH Bethesda, MD 
Javeed A Dhobi, LISB/NIAID/NIH Bethesda, MD 
Nageen Sherani, LISB/NIAID/NIH Bethesda, MD 
Michael G Mage, LISB/NIAID/NIH Bethesda, MD 
Lisa F. Boyd, LISB/NIAID/NIH Bethesda, MD 
David H. Margulies, LISB/NIAID/NIH Bethesda, MD 

Dissecting the specificity of a TCR-mimic antibody targeting TP53 R175H mutation-derived neoantigen

12:40 PM - 1:00 PM 
The emergence of immunotherapy as an important tool in the fight against cancer takes advantage of the exquisite specificity of antibodies. Targets, however, have been limited to those on the cell surface, despite most oncogenic driver mutations occurring in genes which encode intracellular proteins; known as "undruggable" targets. To overcome this limitation, antibodies can be selectively engineered to target mutation-derived neoantigens, peptides derived from mutant proteins that are presented on the cell surface by the Major Histocompatibility Complex Class I (MHC-I), and not their wild-type peptide-MHC counterparts. One such "undruggable" target is tumor-suppressor protein TP53, one of the most commonly mutated driver genes in all cancers. Here, we describe the identification and structural basis of a T cell receptor (TCR)-mimic antibody against the HLA-A*02:01-restricted TP53 R175H epitope, highlighting its specificity. We have developed a TCR-mimic antibody through phage display, selecting the antibody that showed specificity and selectivity toward the mutant peptide TP53 R175H over the wild-type peptide. The TP53 R175H specific antibody was expressed in mammalian cells to generate an optimal protein fold and the antibody Fab fragments were generated using enzymatic cleavage techniques. We performed both structural and biophysical characterization. Structural studies of the Fab/TP53 R175H peptide-MHC complex were carried out using X-ray crystallography to fully understand the interaction between the antibody and TP53 peptide. Furthermore, biophysical characterization included carrying out binding kinetics experiments using Surface Plasmon Resonance (SPR). Determination of the structure revealed the TCR-mimic antibody forms a cage-like configuration around the C-terminal of the TP53 R175H neoantigen, trapping residue H175 into an exposed position by providing a stable interaction. Specifically, all CDRs of the variable heavy chain interact with the displayed TP53 R175H neoantigen. In contrast, only the third CDR of the variable light chain contributes to peptide interaction. Interestingly, the TCR-mimic antibody interaction employs a non-canonical parallel binding mode, different from the typical diagonal orientation utilized by most TCRs and other TCR-mimics. It is possible this new mode of binding contributes to the observed antibody specificity and measured affinity (KD = 86 nM). Exploitation of our detailed structural understanding of the mechanisms of specificity is essential for the development of more potent and selective antibodies. The exquisite selectivity and specificity achievable with antibodies provides the added benefit of distinguishing between wild-type and mutant proteins-the foundation for developing effective treatments with minimal adverse effects to patients. Our study provides a new immunotherapeutic approach to treat cancers with "undruggable" driver alterations. 

View Proposal 226

Author

Katharine Wright

Additional Author(s)

Emily Han-Chung Hsiue, Sidney Kimmel Comprehensive Cancer Center Baltimore, MD 
Jacqueline Douglass, Sidney Kimmel Comprehensive Cancer Center Baltimore, MD 
Michael S. Hwang, Sidney Kimmel Comprehensive Cancer Center Baltimore, MD 
Brian J Mog, Sidney Kimmel Comprehensive Cancer Center Baltimore, MD 
Alexander H. Pearlman, Sidney Kimmel Comprehensive Cancer Center Baltimore, MD 
Suman Paul, Sidney Kimmel Comprehensive Cancer Center Baltimore, MD 
Sarah DiNapoli, Sidney Kimmel Comprehensive Cancer Center Baltimore, MD 
P. Aitana Azurmendi, Department of Biophysics and Biophysical Chemistry Baltimore, MD 
Drew M. Pardoll, Sidney Kimmel Comprehensive Cancer Center Baltimore, MD 
Chetan Bettegowda, Sidney Kimmel Comprehensive Cancer Center Baltimore, MD 
Nickolas Papadopoulos, Sidney Kimmel Comprehensive Cancer Center Baltimore, MD 
Kenneth W. Kinzler, Sidney Kimmel Comprehensive Cancer Center Baltimore, MD 
Bert Vogelstein, Sidney Kimmel Comprehensive Cancer Center Baltimore, MD 
Sandra Gabelli, Johns Hopkins University Ellicott City, MD 
Shibin Zhou, Sidney Kimmel Comprehensive Cancer Center Baltimore, MD 

Coffee Break

1:00 PM - 1:20 PM 

Structural Basis of the Interaction Between Ubiquitin Specific Protease 7 and Enhancer of Zeste Homolog 2

1:20 PM - 1:40 PM 
Anna Bojagora, Varvara Gagarina, Ira Kay Lacdao, Niharika Luthra, Roland Pfoh, Sadaf Mohseni, Danica Chaharlangi, Nadine Tan and Vivian Saridakis Department of Biology, York University, 4700 Keele Street, Toronto, Ontario, M3J1P3, Canada The deubiquitinating enzyme, USP7, regulates the turnover of proteins involved in many diverse cellular processes, including maintenance of genome stability, tumour suppression, epigenetics, DNA replication, cell division, and immune response. Previously defined USP7 substrates, including GMPS, UHRF1, and ICP0, were characterized to interact with the C-terminal domain of USP7 via a KxxxK motif. We identified a common motif in Enhancer of Zeste 2 (EZH2), a histone methyltransferase and the catalytic component of the Polycomb Repressive Complex 2 (PRC2). PRC2 is responsible for methylating Histone 3 Lys27 (H3K27) leading to transcriptional repression of genes involved in differentiation and development. The interaction between USP7 and EZH2 was demonstrated by GST pull-down and co-immunoprecipitation experiments. We co-crystallized Ubl123 of USP7 with an EZH2 peptide containing the predicted interaction site and characterized the structural basis of the interaction, identifying the key residues involved. Mutagenesis studies of these residues demonstrated abolished binding between USP7 and EZH2. The functional relationship between USP7 and EZH2 was investigated using USP7 knock-down and knock-out experiments which corresponded with reduced EZH2 levels in HCT116 carcinoma cells and decreased H3K27Me3 levels in HCT116 USP7 knockout cells respectively. These findings reveal that USP7 regulates both the stability and function of EZH2. 

View Proposal 304

Author

Anna Bojagora, York University

Insights into the molecular mechanism of metal transport by NRAMP family transporters

1:40 PM - 2:00 PM 
Transition metal ions like Mn2+, Fe2+, Ni2+, Co2+, Cu2+, and Zn2+ are essential nutrients that play a crucial role in various metabolic processes in all living cells. Excess or deficiency of these metal ions leads to harmful diseases including cancer, anemia and immune deficiencies. Cellular levels of essential metal micronutrients are thus tightly controlled and regulated. NRAMPs (natural resistance-associated macrophage proteins) are a class of transition metal transporters present in all domains of life that regulate the levels of these essential divalent metal ions within cells and prevent human disorders related to metal insufficiency (like anemia) or overload. In humans, there are two NRAMPs. NRAMP1 is expressed in macrophages and assists in immune response by preventing microbial access to Mn2+ and Fe2+ within pathogen-infected phagosomes. NRAMP2 is widely expressed at low levels in the endosomes of all nucleated cells, and also at the apical surface of epithelial cells in the intestinal tract, where it is responsible for uptake of non-heme Fe2+ from the diet. NRAMP-mediated metal ion transport typically involves co-transported protons. A few structures of bacterial NRAMPs are reported to date which reveal the overall fold (similar to LeuT permease) and provide a preliminary understanding of metal binding and proton co-transport. To obtain a better picture of metal ion selectivity, we obtained additional high-resolution crystal structures in multiple conformational states in apo and metal-bound forms of a bacterial NRAMP from Deinococcus radiodurans (DraNRAMP). To complement the structural data, we performed binding studies using ITC and proteoliposome-based transport assays to understand the thermodynamic parameters associated with metal binding and transport. Overall, the high-resolution structures of DraNRAMP provide a first look at the detailed coordination geometry for the bound metals in the transporter's metal-binding site with insights into metal selectivity determinants. 

View Proposal 132

Author

Shamayeeta Ray, Harvard University Brighton, MA 

Additional Author

Rachelle Gaudet, Professor Cambridge, MA 

Structure of V-ATPase from mammalian brain

2:00 PM - 2:20 PM 
Signal propagation across the chemical synapse between the axon terminal of a presynaptic neuron and the dendrite of a postsynaptic neuron requires the regulated release of neurotransmitters from vesicles into the cleft. The synaptic vesicle membrane is energized by the proton pumping activity of vesicular- or vacuolar-type ATPases (V-ATPases) to allow transporters to load vesicles with neurotransmitters. In V-ATPases, ATP hydrolysis in the catalytic V1 region drives rotation of a central rotor subcomplex and leads to proton translocation through the membrane-embedded VO region. V-ATPase activity is regulated by reversible separation of the V1 and VO¬ regions, with ATP hydrolysis inhibited in the isolated V1 complex and the VO complex becoming impermeable to protons. Fusion of synaptic vesicles with the presynaptic membrane requires separation of the V1 and VO regions but it is not known how these events are coordinated. The mammalian V1 region contains subunits A3B3CDE3FG3H, and the VO region is thought to be composed of acxcde as well as ATP6AP1, also known as Ac45, and ATP6AP2, also known as the (pro)renin receptor. ATP6AP2/PRR is involved in several signalling pathways including the renin-angiotensin system for regulating blood pressure and electrolyte balance and Wnt signalling in stem cells and embryo development. The precise arrangement of subunits in the mammalian VO region remains unclear. Further, mammals have multiple isoforms of some subunits in both V1 and VO that are expressed in a tissue-dependent and cellular-compartment-dependent way, complicating their analysis. We isolated rat synaptic vesicle V-ATPase from rat-brain membranes through its interaction with SidK, a Legionella pneumophila effector protein. CryoEM allowed construction of an atomic model, revealing the mammalian V-ATPase architecture and composition, and defining the enzyme's ATP:H+ ratio as 3:10. The c-ring encloses the transmembrane anchors for cleaved ATP6AP1/Ac45 and ATP6AP2/PRR. The structure shows how ATP6AP1/Ac45 and ATP6AP2/PRR enable assembly of the enzyme's catalytic and membrane regions. 

View Proposal 318

Author

Yazan Abbas Toronto

A novel form of allosteric regulation in an ancient enzyme: mapping GTP’s effect on ribonucleotide reductase with SAXS and crystallography

2:20 PM - 2:40 PM 
Enzyme regulation is crucial to proper function, and the mechanisms that dictate this regulation often require allosteric transitions involving dynamic conformational change. A paradigm of complex regulation is the ribonucleotide reductase (RNR) family of enzymes, which uses a conserved, radical-based mechanism to catalyze the de novo conversion of ribonucleotides to deoxyribonucleotides. In previous work, we elucidated how the RNR of Bacillus subtilis maintains DNA metabolic homeostasis via an unprecedented regulatory mechanism in which active tetrameric complexes interconvert with inhibited filaments. Further work has since uncovered that B. subtilis has evolved yet another "tuning dial" that may be linked to the organism's stress response. SAXS nucleotide titrations and chromatography-coupled SAXS experiments were used to show that the nucleotide GTP reverses RNR inhibition by breaking down inhibited filaments. Crystal structures in turn reveal a novel GTP-binding site and further suggest the mechanism of activation. This new GTP-binding site represents the surprising genesis of not just a new allosteric site but a new allosteric activator among all RNRs, and in doing so provides an exemplar of how evolutionary pressure can rapidly create novel allosteric properties. 

View Proposal 152

Author

William Thomas, Cornell University Ithaca, NY 

Additional Author(s)

Audrey Burnim, Cornell University Ithaca, NY 
Darren Xu, Cornell University Ithaca, NY 
Nozomi Ando, Cornell University Ithaca, NY 

Investigation of ATP-induced global motions of the human DNA mismatch repair sensor complex using time-lapse crystallography 

2:40 PM - 3:00 PM 
Over 1 in 300 people in the US have Lynch syndrome (hereditary nonpolyposis colorectal cancer, HNPCC), which is caused by mutations in DNA mismatch repair (MMR) genes. Over 40% of the Lynch syndrome mutations are found in the human MSH2 gene, which is the common subunit that forms the key MMR lesion sensors MutSα (MSH2-MSH6) and MutSβ (MSH2-MSH3). MSH2 mutations are also identified in over ten other cancer types. The MutS proteins are members of the ABC ATPase family that undergo large conformational rearrangements upon binding/hydrolysis of ADP/ATP. Here we present our time-lapse X-ray crystallography studies to demonstrate the conformational changes of MSH2 as it transitions from ATP to ADP bound states. A dramatic and global conformational rearrangement is triggered after ATP hydrolysis, which spans over 150 Å and causes over 60-degree domain rotations. Time-lapse crystallographic results reveal a network of interactions that link the ABC ATPase center to the DNA binding domains. Moreover, a nascent globular domain formed only in specific nucleotide states has been identified, which is stabilized by residues that are variants of unknown significance in Lynch syndrome and other cancers. Our findings provide new insights into the mechanism of MMR and could aid the development of better colorectal cancer risk prediction models and personalized therapeutic strategies. These results may also extend the understanding of the mechanism of other ABC ATPase family proteins. 

View Proposal 322

Author

Yuqian Shi

Additional Author(s)

You Wang, Duke University Durham, NC 
Lorena Beese, Duke University Medical Center