Hot Structures

Conference: 2022: 72nd ACA Annual Meeting
08/01/2022: 2:00 PM - 5:00 PM
Oral Session 
Portland Marriott Downtown Waterfront 
Room: Salons G-I 


This session will be comprised of talks describing exciting new macromolecular structures. Talks focusing on structures will be highlighted from across all disciplines of structural biology (Cryo-EM, X-ray Crystallography, NMR, SAXS, etc.). The majority of the talks will be selected from submitted abstracts


Structural investigations of arginyltransferases

Arginyltransferases (ATE1s) are a class of essential eukaryotic enzymes that catalyze arginylation, the post-translational transfer of Arg from an aminoacylated tRNA to a range of protein targets. This modification typically occurs at N-terminal acidic residues, though ATE1 can also covalently attach Arg to mid-chain residues. Arginylation may have either degradative or non-degradative effects. For example, some arginylated proteins are processed through the Arg N-degron pathway, which marks these post-translationally modified proteins for degradation by the ubiquitin-proteasome system. In contrast, arginylation may also manifest non-degradative effects such as thermodynamic stability, subcellular relegalization, or functional changes. The diversity of ATE1's targets confers its role as a global regulator, influencing functions such as cardiovascular development, neurological processing, and even the stress response. However, a lack of ATE1 structural knowledge has limited the determination of its three-dimensional fold, how it is regulated, and how it recognizes its substrates. Using a combination of X-ray crystallography, cryo-EM, and size-exclusion chromatography-coupled small angle X-ray scattering (SEC-SAXS), our lab has successfully solved the structure of Saccharomyces cerevisiae ATE1 (ScATE1). The three-dimensional structure of ScATE1 reveals a bilobed protein containing a canonical GCN5-related N-acetyltransferase (GNAT) fold. Structural superpositions and electrostatic analyses indicate this domain as the location of catalytic activity and tRNA binding. Furthermore, the structure reveals the spatial connectivity of the N-terminal domain, which we previously showed binds an [Fe-S] cluster, to the enzymatic active site, hinting at the cluster's regulatory influence. As the first atomic-level structure of any ATE1, this achievement brings us closer to answering pressing questions regarding the molecular-level mechanism of eukaryotic post-translational arginylation. 

View Abstract 1230


Verna Van, University of Maryland, Baltimore County (UMBC) Dept. of Chemistry Halethorpe, MD 

Additional Author(s)

Nna-Emeka Ejimogu, UMBC Baltimore, MD 
Aaron Smith, UMBC Baltimore, MD 

Structural and Bioinformatic Analysis of an Ancient Enzyme Family

Ribonucleotide reductases (RNRs) convert the precursor building blocks of RNA to the precursor building blocks of DNA and are found in every organism that synthesizes its DNA de novo. Because of its potential role in the transition from the hypothesized RNA world to the modern DNA world, understanding the diversity and evolution of this enzyme family has been of great interest. In our study, we have reconstructed the largest RNR phylogeny to-date that unifies all known classes of the enzyme. Surprisingly, our dataset has revealed a small, phylogenetically distinct clade, which we denote as class Ø, placed as a clade diverging near the root of the tree. Using small-angle X-ray scattering (SAXS), cryo-electron microscopy (cryo-EM), and AlphaFold2, we show that the class Ø enzyme is the most minimal RNR structurally characterized to-date. Combined with our phylogenetic analysis, our structural data give insight into how a minimal RNR scaffold resembles the ancestor to the modern RNRs of the aerobic world. 

View Abstract 933


Audrey Burnim, Cornell University Ithaca, NY 

Additional Author(s)

Matthew Spence, Austrailian National University Canberra
Darren Xu, Cornell University Ithaca, NY 
Colin Jackson, Research School of Chemistry Canberra, Australia 
Nozomi Ando, Cornell University Ithaca, NY 

Structures and mechanism of a type IIS restriction endonuclease: PaqCI

Restriction endonucleases are an essential component of innate, 'preprogrammed' phage restriction systems that protect bacteria from foreign DNA. Type II restriction endonucleases are invaluable tools in research because of their ability to identify and cleave specific DNA sequences with extremely high fidelity, as well as their unique mechanisms of cleavage. Type IIS restriction endonucleases contain separate DNA recognition and cleavage domains and recognize asymmetric targets to cleave at exact distances outside of their target sequences. Type IIS restriction endonucleases are employed for a variety of biotech purposes, including the creation of gene-targeting zinc finger and TAL effector nucleases and DNA processing for genome mapping and deep sequencing applications. The most well-studied type IIS enzyme, FokI, has been shown to require multimerization and engagement with multiple DNA targets for optimal cleavage activity; however, details of how it or related enzymes forms a DNA-bound reaction complex have not been described at atomic resolution. Here we describe a series of crystallographic and CryoEM structures in the presence and absence of bound DNA targets that reveal aspects of DNA recognition and cleavage by the type IIS PaqCI restriction endonuclease. The structures illustrate the enzyme's tetrameric domain organization in the absence of bound substrate and the subsequent formation of a tetrameric reaction complex (involving significant domain rearrangements and reformation of the dimer complex) poised to deliver the first of a series of double strand cleavage events. Understanding the diversity of form and function within type II restriction endonucleases by investigating members such as PaqCI can reveal the differences between (1) catalytic architectures and cleavage mechanisms, (2) DNA binding domains and recognition mechanisms, and (3) the transient formation of higher order oligomeric assemblages to help ensure discrimination between self- and non-self. Additionally, PaqCI could be used to create novel artificial enzymes with new specificities that could be used in biotech applications. 

View Abstract 1166


Madison Kennedy, University of Washington Seattle, WA 

Additional Author

Barry Stoddard, Basic Sciences, Fred Hutchinson Cancer Center / Seattle, WA Seattle, WA 

Coffee Break

We invite all of our attendees to join us in the exhibit hall for complimentary coffee during the session break.  

Structure of a HIV-1 IN-Allosteric Inhibitor Complex at 2.93 Å Resolution: Routes to Inhibitor Optimization

HIV integrase (IN) inserts viral DNA into the host genome and is the target of the strand transfer inhibitors (STIs), a class of small molecules currently in clinical use. Another potent class of antivirals is the allosteric inhibitors of integrase, or ALLINIs. ALLINIs promote IN aggregation by stabilizing an interaction between the catalytic core domain (CCD) and carboxy-terminal domain (CTD) that undermines viral particle formation in late replication. Ongoing challenges with inhibitor potency, toxicity, and viral resistance motivate research to understand their mechanism. Here, we present a 2.93 Å resolution structure of a minimal ternary complex formed by the IN CCD, CTD, and the preclinical lead ALLINI BI-224436. The structure reveals side chain orientations and a more precise view of the molecular interactions that underlie ALLINI-induced aggregation of HIV IN. The complex has a pronounced asymmetry, with non-identical ALLINI binding interfaces that depend on the nature of the proximal CTD dimers formed in the crystal lattice. We identify several new interactions including a network of cation-π and π-π interactions at the protein-protein and protein-drug interfaces. An accessible pocket adjacent to the bound ALLINI is occupied by ethylene glycol, suggesting specific directions for drug design. The minimal CCD•ALLINI•CTD assembly, like the full-length protein complex, favors the formation of drug-induced polymers in solution via two modes of CTD dimerization, providing orthogonal evidence supporting a branched polymer mechanism of aggregation. From this improved atomic model, we can generalize the mode of action for first-generation molecules and current clinical leads, facilitating routes for improvement of existing ALLINI scaffolds. We have further extended these studies by determining the structure of another ALLINI, BI-D, and two additional ALLINI-resistant forms of intact IN that are found in replication-competent viruses (INW131C and INN222K), at 4.5 Å. These data reveal structural perturbations that would undermine the branched polymer network promoted by ALLINI binding. Together, these results provide important insights to help optimize ALLINI design. 

View Abstract 909


Kushol Gupta, Biochemistry & Biophysics, Univ of Pennsylvania Philadelphia, PA 

Additional Author(s)

Grant Eilers, Perelman School of Medicine, University of Pennsylvania Philadelphia, PA 
Audrey Allen, Perelman School of Medicine at the University of Pennsylvania Philadelphia, PA 
Saira Montermoso, University of Pennsylvania Philadelphia, PA 
Hemma Murali, Perelman School of Medicine at the University of Pennsylvania Philadelphia, PA 
Robert Sharp, Perelman School of Medicine at the University of Pennsylvania Philadelphia, PA 
Young Hwang, Perelman School of Medicine at the University of Pennsylvania Philadelphia, PA 
Frederic Bushman, Perelman School of Medicine at the University of Pennsylvania Philadelphia, PA 
Gregory Van Duyne, Perelman School of Medicine at the University of Pennsylvania Philadelphia, PA 

Activated dioxygen intermediates at the copper-active site of a lytic polysaccharide monooxygenase

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that oxidize the glycosidic bond of polysaccharides. Since their discovery in 2010 LPMOs have been intensely studied by structural, spectroscopic, and biochemical methods however their catalytic mechanism remains to be established. Experimental data to unambiguously identify the activated copper-oxygen intermediates and their mechanism of formation are especially required to settle the early steps. We will present how we used neutron crystallography to investigate the initial steps of oxygen activation directly following active site copper reduction in Neurospora crassa LPMO9D. Cryo neutron crystallography allowed us to capture an activated dioxygen intermediate, identify its chemical nature, and reveal a conserved second coordination shell residue as a proton donor essential in intermediate formation. The chemical insights from the neutron crystal structure were supported by density functional theory (DFT) calculations and by mining minima free energy calculations. 

View Abstract 885


Flora Meilleur

CryoEM structure of a multivalent ubiquitin ligase complex

Protein ubiquitination is a common posttranslational modification with central roles in eukaryotic cellular physiology. The selection of targets for modification is largely determined by the E3 ubiquitin ligases, which catalyze the transfer of ubiquitin by positioning substrates next to activated E2~ubiquitin conjugates. Of the over 600 known E3 ubiquitin ligases, the largest subclass are the Cullin3-Ring-Ligases (CRL3) with over 70 members. CRL3s are modular assemblies that involve multiple components, including BTB domain substrate binders. These binders usually combine a N-terminal BTB Cul3-binding domain and a C-terminal substrate-binding domain within a single polypeptide. Notably, BTB domains can self-associate into stable dimers, pentamers and oligomers and thus drive the multimerization of CRL3 complexes.

We have identified interactions between KCTD5, a pentameric CRL3 BTB adaptor protein, and several G-protein heterodimers. This raises the possibility that members of the CRL3 E3 ligase family regulate G-protein signalling by targeted ubiquitination. We demonstrate the direct, non-exclusive binding of both Gßγ heterodimers and the Cul3 N-terminal domain with KCTD5 and determined the cryo-EM structure of a 560 kDa 5:5:5 KCTD5:Gßγ:Cul3 complex to a resolution of 3.0 Å resolution. The 15-chain assembly has pseudo-C5 symmetry with large scale dynamics involving rotations of over 40° between the KCTD5/Gßγ and KCTD5/Cul3 moieties of the complex. Modeling a full-length Cul3/Rbx1/E2~ubiquitin assembly into the complex reveals that one particular rotamer positions Gßγ within ~5 Å of the E2~Ub thioester bond. Previously described E3/substrate structures were monovalent and involved flexible peptide substrates. The KCTD5/Gßγ/Cul3 complex presented here demonstrates the role of multivalency in the CRL3 ligases and reveals how the architecture of an E3 ligase can position a structured target for ubiquitination. 

View Abstract 1134


Gilbert Privé, Princess Margaret Cancer Centre, Univ Health Network Toronto

Additional Author(s)

Duc Minh Nguyen, University of Toronto
Alan Ji, University of Toronto
Darren Yong, University of Toronto
Andrew Zhai, University of Toronto
Neil Pomroy, Princess Margaret Cancer Centre
Douglas Kuntz, Princess Margaret Cancer Centre
Darlaine Pétrain, McGill University
Dominic Devost, McGill University
Terry Hébert, McGill University