Poster Session III

Conference: 2020: 70th ACA Annual Meeting
08/05/2020: 4:00 PM  - 5:00 PM 
PS3 
Poster Session 
Virtual  

Description

The ACA holds three evening poster sessions. Poster sessions are organized by the Poster Chairs and feature presentations covering a range of crystallography topics. Poster presentations may not seem as prominent as oral presentations, but they offer a terrific opportunity to interact with other scientists in your field in a structured way.

Presentations

Structural Studies of TraG, the Conjugative Entry Exclusion Protein from the F-plasmid

The F-plasmid of Escherichia coli is representative of conjugative type IV secretion systems for the transmission of mobile DNA elements in bacteria, a significant contributor to the evolution of antibiotic resistance. One of the largest proteins of this system, TraG consists of a membrane-bound N-terminal domain, and a periplasmic C-terminal domain denoted TraG*. Each domain has its own function; the membrane bound N-terminal domain is involved in F-pilus assembly while TraG* is bifunctional. In the donor cell, it interacts with TraN within the outer membrane to facilitate mating pair stabilisation. However, TraG* is also essential in preventing redundant DNA transfer through its interaction with a cognate TraS in the inner membrane of the recipient cell when the recipient carries the same plasmid. Thermofluor, circular dichroism and HDX-MS experiments showed N-terminal truncation mutants of TraG* displayed higher stability and less disordered content relative to full-length TraG*. The 45 N-terminal residues of TraG* were predicted to be highly dynamic, possibly serving as a flexible linker between two independently functioning domains. Further truncation mutants of TraG* were designed to enable protein crystallisation. 

View Proposal 128

Author

Nicholas Bragagnolo, York University Bolton, ON 

Additional Author

Gerald Audette, Dept of Chemistry, York Univ Toronto, ON 

Further evidence supporting the allosteric regulation of PEPCK by anions.

Phosphoenolpyruvate carboxykinase (PEPCK) primarily catalyzes the conversion of oxaloacetic acid to phosphoenolpyruvate as one of the key steps of gluconeogenesis. However, PEPCK is known to be thermodynamically reversible in vitro, catalyzing the reverse of the gluconeogenic reaction, albeit with a lower enzyme activity. These two seemingly conflicting pieces of data pointed towards a hypothesis of kinetic inhibition of PEPCK in vivo. Previous research was presented that elucidated a potential 'reverse-direction' specific mechanism of inhibition of PEPCK. Sufficient evidence was provided for the presence of an allosteric site in PEPCK, leading to inhibition of catalysis in the reverse direction while catalytic function in the 'forward' direction was largely unaffected. Structural and kinetic data supported the model that inhibition in the form of small anions, such as chloride, binding to the allosteric site contributed to the observed unidirectionality of the PEPCK-catalyzed reaction in vivo. Previous research implemented a novel use of anomalous diffraction data to support this hypothesis. PEPCK crystals were soaked over a range of increasing iodide concentrations from 10 to 500 mM (as a proxy for chloride) and the corresponding anomalous signals located at the allosteric site were found to titrate over the range of iodide. Other iodide binding sites including the active site were not found to titrate, and instead no relationship between iodide concentration and anomalous signal could be determined. Binding isotherms were generated for the various binding sites and the resulting binding constant determined for the allosteric site was comparable to the competitive inhibition constant from the kinetic data using chloride. Expanding upon these initial data we have undertaken additional studies to support the allosteric model of anion regulation of PEPCK that include the creation of allosteric site mutant PEPCK forms. Structural and kinetic data collected on these mutants similar to that which was previously collected on the WT enzyme provide further support for our model of directional allosteric regulation of PEPCK by monovalent anions. To further support the model of allosteric regulation we are undertaking studies to directly determine the binding of chloride ions to PEPCK rather than using iodide as a proxy by collecting anomalous diffraction data at long wavelengths. 

View Proposal 155

Author

Sarah Barwell, University of Waterloo Waterloo, ON 

Additional Author

Todd Holyoak, Biology Dept, Univ of Waterloo

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

Contributing authors: 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 288

Author

Bronwyn Lyons, University of British Columbia Vancouver, BC 

Native SAD data collection using microbeam at NSLSII

[i]Ab-initio[/i] single-wavelength anomalous dispersion (SAD) determination of macromolecular structures requires precise measurement of anomalous signal from atoms present in the sample such as S, Mn, P or introduced as heavy atoms. Today, availability of synchrotron microbeams and tunability at low-energy has increased the accessibility for measuring those data sets. We described a native-SAD experiment collecting multiple data sets from a single crystal at an energy of 6550 eV (1.89 Å) by offsetting the starting angle by 45° with a beam size of 5 (v) x 7 (h) and an attenuated flux of 5.85e10 ph/s at the NSLS-II FMX beamline. We used chymotrypsinogen crystals with approximate dimensions of 20 x 30 x 140 μm, which contain one molecule of 245 amino acids in one asymmetric unit. Hence the asymmetric unit contains 10 cysteine residues in disulfide bond pairs and 2 methionine residues for a total 12 sulfur anomalous scattering atoms which result in a calculated Bijvoet fraction of 1.08%. Five 360° data sets were collected per crystal. Specifically, the data was collected using exposures of 0.02 seconds per 0.2° for 360° in a vector of about 138 μm in length on an EIGER 16M detector. For the consecutive data sets, the crystal was rotated by 45° and data was collected again with the same vector until the crystal showed apparent radiation damage. The average radiation dose estimated by RADDOSE was 1.63 MGy per data set. The data was merged with BLEND and the structure was determined [i]ab-initio[/i] using CRANK, the automated package for structure solution via experimental phasing. At each step, the specific programs used are SHELXC/D for the heavy atom location, Solomon for density modification and hand determination, PARROT for density modification, Buccaneer for automated model building and Refmac5 for refinement. The work reported here with multiple data collections from the same crystal with low dose is an alternative to merging data from multiple crystals to obtain highly redundant data sets while balancing maximum signal and minimal absorption. 

View Proposal 231

Author

Kim Phan

Additional Author(s)

Wuxian Shi, Brookhaven National Laboratory
Tao-Hsin Chang
Tihitina Aytenfisu, Johns Hopkins University School of Medicine Baltimore, MD 
Alexei Soares, Brookhaven National Laboratory NY 
Jean Jakoncic, Brookhaven National Laboratory NY 
Andres Hernandez de la Peña, Johns Hopkins University School of Medicine Baltimore, MD 
L. Mario Amzel, Johns Hopkins University School of Medicine Baltimore, MD 
Sandra Gabelli, Johns Hopkins University Ellicott City, MD 

B-factor analysis suggests that L-lysine and R, R-bisLysine allosterically inhibit Cj.DHDPS Enzyme by decreasing its protein dynamics

In Campylobacter jejuni (Cj.) , dihydrodipicolinate synthase (DHDPS) catalyzes the condensation of pyruvate (pyr) and (S)-aspartate-β-semi-aldehyde (ASA) to form dihydrodipicolinate. Cj.DHDPS regulates an essential step in the biosynthesis of L-lysine (Lys) and meso-diaminopimelate in bacterial cell wall synthesis. In our study, the normalized B-factors calculated from crystallographic data suggested that Lys and synthetic bisLys inhibitors allosterically inhibit the enzyme by reducing mobility in the regions that undergo transient conformational fluctuations to facilitate the diffusion of substrate Pyr in the active site for catalysis, consistent with published HDX-MS dynamics studies. The normalized B-factors also showed that bisLys allosterically inhibits DHDPS Y110F lysine insensitive mutant by rigidifying these flexible regions. The B-factor analysis suggest that flexibility in the regions (β8, α9, α10, α11, L20, and α12) may be vital for substrate turn over and catalysis. Normalized B-factors of DHDPS and its Y110F mutant with and without inhibitors in different space groups showed similar flexibility patterns, suggesting the reliability of our B-factors analysis. 

View Proposal 404

Author

Sagar saran

Structural basis of reiterative transcription from the pyrG and pyrBI promoters by bacterial RNA polymerase

Reiterative transcription is a non-canonical form of RNA synthesis by RNA polymerase in which a ribonucleotide specified by a single base in the DNA template is repetitively added to the nascent RNA transcript. We previously determined the X-ray crystal structure of the bacterial RNA polymerase engaged in reiterative transcription from the pyrG promoter, which contains eight poly-G RNA bases synthesized using three C bases in the DNA as a template and extends RNA without displacement of the promoter recognition σ factor from the core enzyme. In this study, we determined a series of transcript initiation complex structures from the pyrG promoter using soak–trigger–freeze X-ray crystallography. We also performed biochemical assays to monitor template DNA translocation during RNA synthesis from the pyrG promoter and in vitro transcription assays to determine the length of poly-G RNA from the pyrG promoter variants. Our study revealed how RNA slips on template DNA and how RNA polymerase and template DNA determine length of reiterative RNA product. Lastly, we determined a structure of a transcript initiation complex at the pyrBI promoter and proposed an alternative mechanism of RNA slippage and extension requiring the σ dissociation from the core enzyme. 

View Proposal 273

Author

Yeonoh Shin, The Pennsylvania State University State College, PA 

Additional Author

Katsuhiko Murakami, Penn State Univ University Park, PA 

Discovery and Characterization of Dual TAF1-ATR Inhibitors

Bromodomain-containing proteins regulate chromatin remodeling and gene transcription through the recognition of acetylated lysines on histones and other proteins. Bromodomain-containing protein TAF1, a subunit of general transcription factor TFIID, initiates preinitiation complex (PIC) formation and cellular transcription. Therefore, TAF1 is a promising target to develop small molecule inhibitors for the treatment of diseases arising from dysregulated transcription such as cancer. Here we report the discovery of a dual bromodomain-kinase inhibitor AZD6738, currently in clinical trial phase I/II as an ATR inhibitor, that binds to the second bromodomain of TAF1 and shows promising selectivity for TAF1 within the bromodomain family. High resolution crystal structures and small-angle X-ray scattering (SAXS) derived solution structures of unliganded bromodomains and inhibitor bound complexes revealed that AZD6738 induces large conformational changes in TAF1 tandem bromodomain leading to the spatial reorientation of the two bromodomains. Parallel investigation with a known TAF1 inhibitor BAY299 also indicated large conformational changes upon ligand binding as well as ligand-induced dimerization of the TAF1 tandem bromodomain. Using the TAF1 inhibitors as chemical probes we showed that bromodomain inhibition of TAF1 activates p53 mediated DNA damage response pathway and prevents cancer cell growth in a p53 dependent manner. Combined, our study provides a structural framework for effective TAF1 inhibition to invoke p53 mediated DNA damage signaling in cancer. 

Author

Md Rezaul Karim Tampa, FL 

Additional Author(s)

Leixiang Yang, Moffitt Cancer Center Tampa, FL 
Jiandong Chen, Moffitt Cancer Center Tampa, FL 
Ernst Schönbrunn, Moffitt Cancer Center Tampa, FL 

Single-particle cryo-EM studies of ERp44-ERAP1 and ERp44-ERAP2 reveal their ER-retention mechanism and structural dynamics involved

ERAP1 is an ER-resident aminopeptidase with roles in antigenic peptide processing for MHC-I presentation and angiotensin processing for blood pressure control. Previous studies had implicated ERp44 C29 in the thioredoxin-like a-domain and ERAP1 C498 in the domain-II "exon-10" loop as important in the ER-retention mechanism. We wanted to investigate interactions between ERp44 and ERAP1 beyond the C29-C498 disulfide that might contribute to the specificity and regulation of complex formation, and to determine whether ERAP2, a paralog of ERAP1 that also generates peptides for antigen presentation, could utilize a similar mechanism for ER retention. Using purified recombinant ERp44 and ERAP1, we confirmed that the C29-C498 intermolecular disulfide forms spontaneously upon mixing ERp44 and ERAP1, resulting in a 1:1 complex. ERp44 interaction with ERAP was not pH-sensitive (5.7 – 7.7), unlike its interaction with other client proteins, suggesting a possible novel mode of interaction. Using cryo-EM we obtained 4.4Ǻ resolution maps for the ERp44-ERAP1 complex. The 3-D volumes contain both closed and semi-open conformations of ERAP1 though, suggesting that the ERp44 interaction might stabilize the less open form of the protein, and reveals substantial domain interactions between the two proteins. We are examining this structure to understand the structural basis for the preferential reactivity of ERp44 C29 over C63 and ERAP1 C498 over C486, and to gain insight into the mechanism of release in the ER. Like ERAP1, ERAP2 is ER-resident, but the retention mechanism is unknown. We observed a similar interaction of ERp44 with ERAP2 as with ERAP1, with ERp44 C29 forming an intermolecular disulfide with ERAP2 C514. Cryo-EM 3D volumes for the ERp44-ERAP2 complex at 6.0Ǻ reveal ERAP2 in mostly open conformation. These results suggest that ERAP2 is retained within ER by a similar mechanism as ERAP1 but in a different conformation as ERAP1. This might propose different conformational dynamics of ERAP2 as ERAP1 on interaction with ERp44. 

View Proposal 337

Author

Richa Arya

Additional Author

Lawrence Stern, UMass Medical School Worcester, MA 

Structural basis for bisphosphonate-mediated inhibition of Leishmania major FPPS

Leishmaniasis, a major parasitic disease caused by infection with parasites of the genus Leishmania, affects about 12 million people in 98 countries. The most common form of leishmaniasis, cutaneous leishmaniasis, is caused by Leishmania major. Nitrogen containing bisphosphonates have been shown to have antiparasitic activity against Leishmania in vitro, by targeting the parasite farnesyl diphosphate synthase (FPPS), an enzyme essential for the promastigote and amastigote stages of Leishmania major. Bisphosphonates represent a compelling alternative for the treatment for leishmaniasis due to their safety in humans compared to current drug treatments, which have limitations because of high toxicity and drug resistance. The X-ray crystallographic structures of complexes of LmFPPS with three bisphosphonate inhibitors and Ca 2+ at 2.25, 2.30 and 1.55 Å resolution provided key information about the interaction with the inhibitors and the protein. The complex of LmFPPS with hydrogen (2-(1-hexyl-1H-imidazol-3-ium-3-yl)-1-phosphonoethyl) phosphonate (1216) displays well resolved electron density for the bisphosphonate as well as for the substrate isopentenyl pyrophosphate (IPP) and three Ca2+ ions. On the other hand, the complex of LmFPPS with the inhibitor hydrogen (1-phosphono-2-(1-propyl-1H-imidazol-3-ium-3-yl)ethyl)phosphonate (1337), shows clear electron density for the bisphosphonate, a pyrophosphate molecule and three Ca2+ ions without any density for IPP. The crystal of the complex of LmFPPS with the third inhibitor, hydrogen (1-hydroxy-1-phosphono-2-(1-propyl-1H-imidazol-3-ium-3-yl)ethyl)phosphonate (1336), shows clear electron density for the bisphosphonate and three Ca2+ ions with no electron density for IPP and a pyrophosphate. Comparison of the LmFPPS-bisphosphonate-Ca2+ complex structures show that the bisphosphonates that lack a hydroxyl at the geminal carbon C1 align well with each other (rmsd 0.25 Å). The presence of OH at the C1 carbon in compound 1336, results in a displacement of the calcium atoms by 0.6-0.8 Å. Moreover, Glu-98, a residue of the conserved first aspartate rich motif which is typically a ligand of the divalent cations, is also at hydrogen bonding distance of the 1336-OH group. Though the conformation of the active site residues is very similar in all the structures, Phe-94 in the LmFPPS-1216 structure faces away from the binding pocket of the bisphosphonate aliphatic chain. Interestingly, binding kinetics determined by SPR shows that compound 1216, 3 carbons longer than 1337, has a four-fold higher affinity (KD=120 nM) than the 1337 compound. Detailed analyses of the crystal structures, binding affinities and cellular activity provide insights into bisphosphonate design for effective LmFPPS inhibition. 

View Proposal 398

Author

P. Aitana Azurmendi, Department of Biophysics and Biophysical Chemistry Baltimore, MD 

Additional Author(s)

Sweta Maheshwari, Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine Baltimore, MD 
William Hong, Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine Baltimore, MD 
Michael Murphy, Cytiva Marlborough, MA 
Eric Oldfield, Department of Chemistry, University of Illinois at Urbana-Champaign Urbana, IL 
L. Mario Amzel, Johns Hopkins University School of Medicine Baltimore, MD 
Marcelo Sousa Silva, Global Health and Tropical Medicine, Institute of Hygiene and Tropical Medicine Lisbon
Sandra Gabelli, Johns Hopkins University Ellicott City, MD 

Structural characterization of three distinct beta-glucuronidases from the gut commensal bacterium Roseburia hominis

Glycoside hydrolase enzymes encoded by the gut microbiome play a key role in the breakdown of a variety of dietary carbohydrates and glycoconjugates. The benefits of this process are two-fold: the human host expands its metabolic potential and gains dietary energy, and the microbiota gain an energy source from the products of this breakdown. Here, we examine the structure and function of three β-glucuronidase (GUS) glycoside hydrolases from Roseburia hominis, a gut commensal microbe. GUS structures solved using both X-ray crystallography and cryo-EM reveal differences in their tertiary and quaternary structures. RhGUS1 contains two unique loops, one that encloses the active site and another with α-helical structure. Additionally, RhGUS1 is a tetramer, resulting in a solvent-occluded active site. Conversely, RhGUS2 and RhGUS3 are both dimers with solvent-exposed active sites. These GUSs also contain a C-terminal domain and a FMN-binding site in the beta-sandwich region that are not observed in RhGUS1. These structural differences result in differential processing of several endogenous and exogenous glucuronide substrates in vitro. Together, these data indicate that the structural variation of GUS enzymes from Roseburia hominis allows for this gut microbe to process a range of structurally diverse glucuronide substrates. 

View Proposal 126

Author

Morgan Gibbs, UNC at Chapel Hill Carrboro, NC 

Additional Author(s)

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

Discovery of an inhibitor of succinyl-CoA synthetase

Tartryl-CoA was discovered in the crystal structure of human GTP-specific succinyl-CoA synthetase (SCS). SCS catalyzes the only substrate-level phosphorylation of the citric acid cycle. It catalyzes the reversible reaction: succinyl-CoA + NDP + Pi ⇌ succinate + CoA + NTP in the presence of magnesium ions. Humans have two different SCSs, ATP-specific SCS and GTP-specific SCS. The crystallization experiment included GTP-specific SCS, ADP, CoA, and magnesium ions in the protein solution, while polyethylene glycol 3350 and ammonium tartrate were in the well solution. During crystallization, tartryl-CoA was synthesized from tartrate and CoA, and, instead of being released from the enzyme, tartryl-CoA remained as a bound ligand. The CoA portion binds as expected in the CoA-binding site, but the tartryl portion binds in the phosphate-binding site, close to the catalytic histidine residue. Although succinyl-CoA is structurally similar to tartryl-CoA, succinyl-CoA would not bind to SCS in the same way as tartryl-CoA. The two extra hydroxyl groups of tartrate contribute to the binding of tartryl-CoA. Tartryl-CoA acts as an inhibitor, inhibiting SCS after a single turnover. 

View Proposal 385

Author

Ji Huang Calgary

Additional Author

Marie Fraser Caglary

Structure-activity relationship analysis of mosquito glutathione S-transferase Noppera-bo and its inhibitors for insecticide development

[Introduction] A new insecticide targeting mosquitoes is globally needed because mosquitoes are serious vectors of many infectious diseases in the world. In this study, we focused on the insect steroid hormone called ecdysteroid. Ecdysteroid is the principal insect steroid hormone important for regulating developmental transitions such as molting and metamorphosis. Ecdysteroids are biosynthesized from dietary cholesterol through intermediate steps by a series of enzymes. One of the ecdysteroidogenic enzymes, called Noppera-bo (Nobo) (Enya et al. [i]Sci. Rep.[/i] 2014), is a glutathione [i]S[/i] -transferase that catalyzes glutathione conjugation. We focused on Nobo from the yellow fever mosquito [i]Aedes Aegypti[/i] (AeNobo) for insecticide development. [Results] 1. Identification of small compound inhibitors by high throughput screen A high-throughput screen of a small molecule library and further validation analysis were conducted to identify compounds that inhibit the AeNobo enzymatic activity. Finally, we found compounds that have a flavonoid nucleus (tentatively designated AeNICs, standing for AeNobo Inhibitor Compounds) as potent inhibitor compounds, which exhibit IC50 values of less than 10 µM. 2. Identification of binding mode of the inhibitors with AeNobo by X-ray crystallography AeNobo was crystallized and soaked with a solution containing AeNIC. AeNobo structure complexed three AeNICs were determined at 1.46 Å, 1.75 Å, and 1.82 Å respectively. All AeNICs bind the hydrophobic site (H-site) of AeNobo (Figure 1). 3. Analysis of protein dynamics by molecular dynamics simulation (MD) ([i]in silico[/i] analysis) To evaluate the contribution of interaction for binding between AeNICs and AeNobo, MD simulation was conducted. We found that AeNICs consistently interacted with Glu113 of AeNobo (Figure 1). 4. Characterization of structure-activity relationship by in vitro enzyme assay ([i]in vitro[/i] analysis) 4-1 Evaluation of the importance of the hydrogen bond between Glu113 and AeNICs: A point-mutant protein that substitutes Glu113 with alanine (AeNobo[Glu113Ala]) was constructed and utilized for enzymatic assay. We found that the inhibitory activity of AeNICs was decreased against AeNobo[Glu113Ala] compare to that against wild type AeNobo. 4-2 Evaluation of the importance of planar structure: Since AeNICs tend to have a planar chemical structure, we hypothesized that a planar structure of AeNICs might affect inhibitory activity. To test the hypothesis, we compared the inhibitory activity of AeNIC derivative compounds with non-planar structures. We found that the IC50 of such derivatives was more than 25 µM, suggesting the planar structure is essential for inhibitory activity (Table1). 5. mosquitoes larvicidal assay. ([i]in vivo[/i] analysis) To evaluate whether the AeNIC affects mosquito ([i]Aedes aegypti[/i]) development, a larvicidal assay was conducted. Mosquito larvae were reared into water containing 10 ppm AeNIC and observed their development (Table1). Consistent with in vitro assay, 45 % of AeNIC treated mosquito larvae died. In contrast, non-inhibitors such as AeNIC derivative compounds with non-planar structures did not affect mosquito development (Table1). [Conclusion] We found two characteristics of AeNIC inhibitory activity against AeNobo. (1) Interaction with Glu113 via a hydrogen bond and (2) the planar structure is essential for inhibitory activity (Table1). Moreover, AeNICs have an insecticidal effect. We will try the identification of essential amino acid residue for the restriction of binding with AeNobo. 

View Proposal 387

Author

Kazue Inaba, Grad. Sch. of Life and Environ. Sci, Univ. of Tsukuba Tsukuba

Additional Author(s)

Kotaro Koiwai, Structural Biology Research Center, IMSS, KEK
Kana Ebihara, Grad. Sch. of Life and Environ. Sci, Univ. of Tsukuba
Ryunosuke Yoshino, Grad Sch Comp Human Sci, Univ. Tsukuba
Takatsugu Hirokawa, Molprof, AIST
Riyo Imamura, OCDD, Univ. of Tokyo
Hirotatsu Kojima, OCDD, Univ. of Tokyo
Takayoshi Okabe, OCDD, Univ. of Tokyo
Tetsuo Nagano, OCDD, Univ. of Tokyo
Hideshi Inoue, Tokyo Univ. of Pharmacol. Life Sci.
Yuuta Fujikawa, Tokyo Univ. of Pharmacol. Life Sci.
Chisako Sakuma, Ctr. Med. Entomol., Jikei Univ. Sch. Med.
Hirotaka Kanuka, Ctr. Med. Entomol., Jikei Univ. Sch. Med.
Fumiaki Yumoto, Structural Biology Research Center, IMSS, KEK
Toshiya Senda, Structural Bio Research Ctr Inst of Materials Structure , High Energy Accelerator Research Org Tsukuba
Ryusuke Niwa, Life Sci. Center of TARA, Univ. of Tsukuba

Structural basis of Neisserial lactoferrin binding protein B function

Neisseria are exclusive human pathogens causing meningitis, septicemia, and gonorrhea. Neisserial pathogens acquire iron from host proteins such as transferrin, lactoferrin, and hemoglobin using outer membrane protein systems. The lactoferrin binding protein (Lbp) system, composed of integral membrane protein LbpA and lipoprotein LbpB, has been proposed to selectively hijack iron from lactoferrin protein. LbpB also provides protection against host antimicrobial peptide lactoferricin. The molecular mechanisms for Lbp system functions remain unknown. In the current study, we determined the structure of LbpB in complex with lactoferrin. We show that the N-lobe of LbpB interacts with the C-lobe of lactoferrin. Structural alignment analysis indicates virtually no conformational changes upon complex formation. Our structure also provides insight into LbpB's specificity towards holo-lactoferrin over apo-lactoferrin. We show that lactoferrin and lactoferricin binding to LbpB are independent. We propose that LbpB binding locks lactoferrin in an iron-bound state for efficient iron scavenging during Neisserial infections. 

View Proposal 412

Author

Ravi Yadav, Purdue University West Lafayette, IN 

Additional Author(s)

Srinivas Chakravarthy, BioCAT (Sector 18, APS), Illinois Institute of Technology Argonne, IL 
Courtney Daczkowski, Department of Biochemistry, Purdue University West Lafayette, IN 
Nicholas Noinaj, Purdue University

Crystal and Solution structures of Proliferating Cell Nuclear Antigen from Crenarchaeon Aeropyrum pernix

[b]Introduction[/b] Sliding clamps are ring-shaped proteins that encircle DNA and confer high processivity on DNA polymerases. In bacteria, the β-clamp protein forms a homodimer, whereas in eukaryotes or euryarchaeotes, proliferating cell nuclear antigen (PCNA) proteins form homotrimers. However, PCNA from [i]Aeropyrum pernix[/i] ([i]Ap[/i]PCNA), a crenarchaeote species, forms a heterotrimer. The actual structure of [i]Ap[/i]PCNA-mediated sliding clamps and the mechanism by which they slide along DNA is unknown.The present study aimed to analyze the crystal and solution structure of the heterotrimeric ring of [i]Ap[/i]PCNA, examine its interaction with DNA and other proteins, and elucidate the mechanism of PCNA function. Previously, we have analyzed the crystal structure of [i]Ap[/i]PCNA1 from the APE_0162 gene.[sup]1)[/sup] [b]Experimental procedures[/b] Each [i]Ap[/i]PCNA molecule, which constitutes a heterotrimer, was expressed using the [i]Escherichia coli[/i] expression system. The proteins were purified using heat treatment, ammonium sulfate precipitation, and column chromatography. The purified proteins were crystallized using the vapor-diffusion method and the crystals were analyzed by X-ray diffraction. The crystal structures of [i]Ap[/i]PCNA2 from the APE_0441.1 gene and [i]Ap[/i]PCNA3 from the APE_2182 gene were determined by the single-wavelength anomalous dispersion method using platinum. To verify the ring shape of [i]Ap[/i]PCNA2 in solution, the solution structure was analyzed using size-exclusion chromatography-small-angle X-ray scattering (SEC-SAXS). A mixture of [i]Ap[/i]PCNAs was analyzed by SEC-multi-angle light scattering for the presence of a complex, and the solution structure was analyzed by SEC-SAXS. [b]Results[/b] Crystals of [i]Ap[/i]PCNA2 were grown up to 0.1 mm and diffracted to 1.8 A resolution. [i]Ap[/i]PCNA2 crystallized as a tetramer. Two sets of dimers, linked through hydrogen bonds, joined back to back in an asymmetric unit. This structure also formed a tetrameric-ring. Crystals of [i]Ap[/i]PCNA3 diffracted to 1.9 A resolution. The structure formed a trimeric ring. The C-terminal interacted with the cleavage site, a possible PCNA-interacting peptide box (PIP-box) binding site, of the adjacent trimeric ring. The solution structure of the complex was similar to shape of the British Isles islands. [i]Ap[/i]PCNA2 and [i]Ap[/i]PCNA3 interacted in a similar manner as the PCNA rings of other organisms; however, [i]Ap[/i]PCNA1 was located such that it did not form a perfect ring-shaped structure. The scattering curves of the complex and those of the model edited trimeric ring were almost similar with minor differences. [b]Discussion[/b] The crystal structure of [i]Ap[/i]PCNA2 showed that it exists as a tetrameric-ring. PCNA is a trimeric ring. Although this molecule, which forms a trimer, is unstable as a tetramer, the crystal structure forms a tetrameric-ring. Hydrogen bonds present in neighboring molecule, from Thr175 to Glu179 and Glu109 to Ile114, form a β-sheet. This flexible interaction suggests that the protein could be forming tetrameric-ring-shaped structure. The crystal structure of [i]Ap[/i]PCNA3 shows that the C-terminal contacts the cleavage site surrounded by β-sheets, formed behind the α-helices, and the inter-domain connecting loop between the neighboring trimeric-rings. Leu249 in the C-terminal is placed in the hydrophobic pocket formed by Met40, Leu47, Leu131, Pro228, Ala246, and Pro247 (Figure). Thus, this hydrophobic cleavage site may be a binding site for PIP-box motif containing proteins. The N-terminus of [i]Ap[/i]PCNA1 is approximately 10 residues longer than that of [i]Ap[/i]PCNA2 and [i]Ap[/i]PCNA3. This could be why the tripartite complex is not ring shaped. Moreover, Met16 is present downstream of the N-terminal of [i]Ap[/i]PCNA1. In the future, the effect of N-terminus deletion and binding of the DNA duplex on [i]Ap[/i]PCNA1 structure should be evaluated. 1) T. Yamauchi, [i]et al.[/i], Purification and Crystallization of PCNA from thermophilic archaea. Poster presented at: 138[sup]th[/sup] Annual Meeting of the Pharmaceutical Society of Japan; Mar. 25-28, 2018; Kanazawa, JAPAN. 

View Proposal 395

Author

Takahiro Yamauchi, Graduate School of Life Science and Technology, Iryo Sosei University, Japan Iwaki, Fukushima

Additional Author(s)

Tsubasa Takemori, Faculty of Pharmacy, Iryo Sosei University Iwaki, Fukushima 
Makiko Kikuchi, Graduate School of Science and Engineering, Iryo Sosei University Iwaki, Fukushima 
Yasuhito Iizuka, Graduate School of Life Science and Technology, Iryo Sosei University Iwaki, Fukushima 
Satoshi Ishikawa, Faculty of Pharmacy, Iryo Sosei University Iwaki, Fukushima 
Masaru Tsunoda, Graduate School of Life Science and Technology, Iryo Sosei University Iwaki, Fukushima 

A High Pressure Macromolecular Crystallography Capability Developed at CHESS

The Earth's biosphere exists at pressures ranging largely from 100 bar to several kbar, but the pressure effects on organisms in a very significant part of the biosphere remain very little understood. To leverage research in such a significant but unexplored territory, CHESS have recently combined a team effort and developed a crystallography capability, allowing one to measure pressure effects on biomacromolecules at the atomic level. Typically, the instrumentation includes several key components: 1) gas-driven membrane in-vivo DAC cell with an angular opening of 100 degrees; 2) double calibrated in-situ pressure technique with ability of controlling and measuring pressure at a fine step of 50 bars; and 3) synchronized experimental assembly; and 4) in-situ imaging system of small sample in DAC. All the components given above are built in an optical table, which can be rolled-in and rolled out of the ID7B2 hutch. Upon synchronization of the sample in DAC with X-rays and large area detector (e.g. Pilatus 6M), a full set of diffraction images for well resolving the structure can be collected from either a single or multiple crystals. 

View Proposal 386

Author

Xin Huang

Additional Author(s)

Zhongwu Wang, Cornell University Ithaca, NY 
Aaron Finke, Cornell University Groton, NY 
Doletha Szebenyi, MacCHESS, Cornell Univ Ithaca, NY 
Qingqiu Huang, Cornell University Ithaca, NY 
Sol Gruner, Physics Dept. & CHESS, Cornell Univ
Durgesh K Rai, Cornell High Energy Synchrotron Source Ithaca, NY 

Development of a model protein for HIV Tat structural study and drug development

Despite significant advances in antiretroviral treatment, HIV remains a global health issue with over 37.9 million individuals living with HIV in 2018. Treatment is highly effective at controlling the virus but has cytotoxic side effects and must be maintained for life or viral production will resume. Drugs that target additional HIV mechanisms can help overcome issues with resistant strains and provide new avenues for treatment. The 12-kDa HIV Tat protein is a target for the development of new therapeutics. Tat is essential for viral replication to overcome the host RNA promoter proximal pausing defense mechanism. To continue paused transcription, Tat binds to a sequence of RNA in the 5' LTR of the HIV genome called TAR. The Tat:TAR complex recruits a host protein complex consisting of CDK9, AFF4, and CycT1 that rescues paused transcription. Despite extensive efforts to determine the molecular structure of the Tat:TAR complex, there is no structure of the entire C-terminal half of the 101 amino acid Tat protein, including the arginine rich motif (ARM) shown to be crucial for TAR binding. Efforts to target the Tat:TAR interaction through the development of small molecules and RNA mimetics have failed to produce viable drugs. Instead, we are targeting the CycT1:Tat interaction by using existing structural data to computationally design a small molecule inhibitor of the complex. Unfortunately, structural biology and structure-based drug discovery methods are hindered by the requirement that the protein be expressed in eukaryotic cells, generally insect cells, which reduces the ease of acquiring material. Here, we report the development of a linked chimera model protein consisting of CycT1 linked to the HIV Tat protein. The fusion of the two proteins increases their solubility, and by expressing the chimera protein with a cleavable maltose-binding protein tag the CycT1:Tat complex can be expressed and purified in large quantities from Escherichia coli cells. We determined the crystal structure of this CycT1:Tat protein bound to AFF4 and without RNA in space group P1 to 2.6 Å. As shown, the asymmetric unit contains four CycT1:Tat:AFF4 protomers. The structure matches previously published CycT1:Tat structures, showing a RMSD of 0.421 Å. Using the CycT1-Tat chimera we are able to easily test numerous different lengths of the Tat protein for stability and RNA binding and subsequently determine the structure of Tat, containing the ARM, complexed with RNA. Additionally, our chimera protein will allow us to optimize our computationally designed inhibitor through structure-based drug design methods. 

View Proposal 159

Author

Joshua Rose, National Cancer Institute Rockville, MD 

Additional Author

Xinhua Ji, National Cancer Institute, NIH Frederick, MD 

Structural analysis of human intestinal alpha-glucosydases: Sucrase-isomaltase and maltase-glucoamylase.

Sucrase-isomaltase (SI) and maltase-glucoamylase (MGAM) both belong to the glycoside hydrolase family 31 (GH31). These two enzymes each contain two catalytic subunits at the N- and C-termini. Each of the subunits possesses the characteristic (β/α)8 barrel catalytic pocket inherent to this family, and all the subunits of these enzymes employ the double displacement mechanism and conserve the two characteristic consensus motifs containing the catalytic residues; the aspartic acid in WIDMNE acts as the catalytic nucleophile while the aspartic acid within the motif HWLGDN functions as the proton donor. While SI and MGAM show high sequence similarity, each subunit of the two enzymes hydrolyzes specific substrates with a level of overlapping activity, presumably to enhance the efficiency of carbohydrate digestion. This suggests that residues outside the catalytic pocket contribute to the substrate affinity. By comparing the structures of the different subunits, the goal of this project is to identify key residues involved in substrate affinity among the four subunits. Initial computational modelling and docking based on structures in hand will be supplemented by new crystallographic or Cryo-EM models of the human enzymes, expressed in yeast cells. 

View Proposal 142

Author

Nardo Nava, University of Waterloo Waterloo

Additional Author

David Rose, Dept of Biology, Univ of Waterloo

Optimal Box Size and Mask Diameter based on Defocus Distribution for Single Particle Cryo-EM

Recently, it has been demonstrated that single particle analysis (SPA) using 200 keV CryoEM paired with direct electron detector (DED) is capable to reconstruct < 200 kDa protein structures at resolution higher than 3.0 Å. However, the majority of near-atomic resolution cryoEM structures has been determined using 300 keV cryoEMs equipped with DEDs. As a consequence, many of typical parameter settings for cryoEM session and image processing steps are based on the accumulated experience of 300 keV cryoEMs, such as defocus range for EM sessions and amplitude contrast for CTF estimation. Therefore, we revised the parameters for 200 keV acceleration voltage, and found out merely optimizing mask diameter and box size based on defocus distribution of dataset can improve the resolution. 

View Proposal 406

Author

Toshio Moriya

The 1.9Å structure of PA5083 – a 116 residue protein with 1 ordered sulfur - determined by Native-SAD using in-house data and phenix.autobuild recycling.

The 1.9Å structure of PA5083 – a 116 residue protein with 1 ordered sulfur - determined by Native-SAD using in-house data and phenix.autobuild recycling. Dayong Zhou1, John P. Rose1 Diana Downs2, and Bi-Cheng Wang1 Departments of 1Biochemistry & Molecular Biology and 2Microbiology, The University of Georgia, Athens, Georgia 30602, USA Abstract PA5083 a Rid2 (Reactive Intermediate Deaminase) protein from Pseudomonas aeruginosa known to have broad imine deaminase activity against iminoarginine has been determined to 1.9Å resolution using Native-SAD. The Rid family enzymes are of interest since they play important roles in nutrition, amino acids biosynthesis, mitochondrial maintenance and other biological processes by reducing the accumulation of toxic metabolite intermediates. The structure determination is noteworthy in that (1) the data were collected in-house ( = 1.5418) and consisted of a single set of 1440 quarter degree images (total rotation 360°, multiplicity 16.6), (2) the 116 residue enzyme has only two sulfur containing residues (Met 1 and Cys 17) giving a Bijvoet ratio 0.374 assuming Met 1 is disordered (it was), (3) the initial results from phenix.autosol gave a "very low" (autosol) FOM of 0.16 with R and Rfree values of 0.5049 and 0.5505 respectively and (4) the structure was built from the initial autosol phases using several rounds of model building with phenix.autobuild. The R and Rfree values for the refined model are 0.200 and 0.233 respectively. Details of the Native (sulfur atom)-SAD analysis and the PA5083 crystal structure will be presented. Work supported in part by funds from the University of Georgia Foundation, and the National Institutes of Health (1S10OD021762-01). Keywords: Native (sulfur atom)-SAD; PA5083; Reactive Intermediate Deaminase; Rid2 Protein; Crystal Structure; Challenging Analysis 

View Proposal 423

Author

Dayong Zhou, University of Georgia

Additional Author(s)

John Rose, SER-CAT/University of Georgia Athens, GA 
Bi-Cheng Wang, University of Georgia
Diana Downs, University of Georgia Athens, GA 

Crystal structure of a bacterial Dicer that is ideal for the preparation of heterogeneous siRNA cocktails

Ribonuclease III (RNase III) represents a highly conserved family of double-stranded RNA (dsRNA) specific endoribonucleases that are important for RNA processing and post-transcriptional control of gene expression. The RNase III family includes prokaryotic RNase III and eukaryotic Rnt1p, Drosha, and Dicer, among which the RNase III from Escherichia coli (EcRNase III) is the most comprehensively studied member. It contains a characteristic RNase III domain (RIIID) and a dsRNA-binding domain (dsRBD) and its E38A mutant (EcE38A) has been utilized to produce small interfering RNAs (siRNAs), mimicking the product of Dicer. The siRNA cocktails thus produced, however, were not heterogeneous due to base-specific substrate selection of the enzyme. Further engineering EcE38A, we created a triple mutant of EcRNase III (EcE38A/E65A/Q165A) that does not have sequence specificity and therefore is ideally suited for producing heterogeneous siRNA cocktails to be used in gene silencing studies. Hence, we refer to this triple mutant as a bacterial Dicer. Here, we present the crystal structures of the bacterial Dicer in complex with products of dsRNA cleavage, showing three slightly different cleavage site arrangements and providing structural basis for its Dicer-like properties. There are two independent molecules in the asymmetric unit. The two molecules dimerize to form a catalytic valley that accommodates dsRNA. The RIIID of each subunit cleaves one RNA strand and the cleavage of both strands generates a 2-nucleotide 3′ overhang, a typical feature of the product of all RNase III enzymes. EcRNase III was discovered in 1968. The structures reported here represent the first three-dimensional structures of the founding member of the RNase III family. 

View Proposal 213

Author

sudhaker dharavath

Nanobodies for isoform-specific study of voltage-gated sodium channels (Navs)

Nav1.4 and Nav1.5 are voltage-gated sodium channels that play an important role in the generation of action potential in excitable tissues. Navs control the passage of sodium ions into cells in response to changes in cellular membrane potential. Myotonia, hyperkalemic periodic paralysis, LongQT syndrome and Brugada syndrome are human genetic diseases caused by dysfunction of Nav proteins that harbor mutations in their C-terminal (CT), cytoplasmic regions. To study the Navs proteins with isoform-specificity, we have selected nanobodies; single, variable heavy-chain only antibody domains (VHH domains, 15kDa) derived from llamas. We generated a nanobody phage library by immunizing a llama with purified CTNav1.4 in complex with Calmodulin (CTNav1.4-CaM). High-affinity nanobody clones to CTNav1.4-CaM complex were selected by immune panning from the phage library and used for periplasmic expression in E. coli. Two anti-CTNav1.4-CaM nanobodies; Nb17 and Nb82 were expressed and purified to homogeneity, while Nb82 was further crystallized and structure determined to 2.0 Å resolution. The asymmetric unit includes four copies with almost identical conformations. The nanobody-fold is decorated by two Π-helices present in CDR1 and CDR3, between β3-β4 and β9-β10 loops. CDR3, the major contributor for antigen recognition and specificity, folds as a random coil that wraps around the β-sheets β6-β5-β4-β9 finishing up in a Π-helix of seven residues. Interestingly, Nb82 stabilizes the CTNav1.4-CaM complex as observed by a 'right-shift' of 13°C in the melt curve of CTNav1.4-CaM, while forming a stable CTNav1.4-CaM+Nb complex on size exclusion chromatography. 

View Proposal 232

Author

Lakshmi SRINIVASAN, Johns Hopkins University Baltimore, MD 

Additional Author(s)

Vanina Alzogaray, Fundacion Instituto Leloir Buenos Aires, Argentina 
Selvakumar Dakshnamurthy, ForteBio Fremont,, CA 
Katharine Wright
Jesse Yoder, IMCA-CAT Lemont, IL 
Sebastian Klinke, Fundacion Instituto Leloir Buenos Aires, Argentina 
Fernando Alberto Goldbaum, Fundacion Instituto Leloir Buenos Aires, Argentina 
L. Mario Amzel, Johns Hopkins University School of Medicine Baltimore, MD 
Sandra Gabelli, Johns Hopkins University Ellicott City, MD 

Structure of an ALS/MSP-related D290V-mutation-containing hexapeptide in hnRNPA2 low-complexity domain reveals differences from its corresponding wild type peptide

Heterogeneous nuclear ribonucleoprotein A2 (hnRNPA2) is an RNA-binding protein involved in RNA transport and stabilization through a membraneless organelle named stress granule. Disruption of stress granules dynamic is associated with neurodegenerative diseases like amyotrophic lateral sclerosis (ALS) and multisystem proteinopathy (MSP). HnRNPA2 contains two RNA recognition motifs and one low-complexity region (LCD). A familial ALS/MSP-related mutation (D290V) within the hnRNPA2 LCD has been identified in previous studies that show evidence of it exacerbating the hnRNPA2 LCD's tendency to assemble into fibrils. We determined the crystal structure of a key hexapeptide (GNYNVF) containing this single point mutation and compared it biochemically to the wild type hexapeptide (GNYNDF). The mutant peptide forms a "steric zipper" motif and the sidechains of the two β-sheets mate tightly with each other in a dry interface. As expected, the mutant peptide is energetically stable and forms a homo-zipper. We then seek to characterize the differences between the wild type and the mutant peptides by electron microscopy. We shook both peptide solutions for a week at room temperature. Due to its energetic stability, mutant peptide forms white precipitates which are composed of wide thick crystals. On the contrary, the wild type peptide forms a hydrogel, which to our knowledge is the shortest segment that forms a hydrogel, and TEM images indicate that the hydrogel is composed of thin fibrils. The wild type peptide exhibits similar physical behavior as the full-length hnRNPA2 LCD, being able to form a hydrogel that is composed of amyloid-like fibrils. This result suggests that the mutant peptide GNYNVF with the pathogenic mutation forms a more stable structure than the wild type peptide GNYNDF, and converts a hydrogel-promoting segment into an aggregation-promoting segment. 

View Proposal 347

Author

David Eisenberg, Univ Of California Los Angeles Los Angeles, CA 

Additional Author(s)

Qin Cao, UCLA-DOE Institute Los Angeles, CA 
Jiahui Lu, UCLA-DOE Institute Los Angeles, CA 
Michael Hughes, UCLA-DOE Institute Los Angeles, CA 
Michael Sawaya
David Boyer, UCLA-DOE Institute Los Angeles, CA 
Duilio Cascio, UCLA-DOE Institute Los Angeles, CA 
David Eisenberg, Univ Of California Los Angeles Los Angeles, CA 

Structure of the human respiratory syncytial virus M2-1 protein in complex with a short positive-sense gene-end RNA

The M2-1 protein of human respiratory syncytial virus (HRSV) is a zinc-binding transcription anti-terminator that regulates the processivity of the HRSV RNA dependent RNA polymerase (RdRP). Here, we reported a crystal structure of HRSV M2-1 bound to a short positive-sense gene-end RNA (SH7) at 2.7 Å resolution. We identified multiple critical residues of M2-1 involved in RNA interaction and examined their roles using mutagenesis and MicroScale Thermophoresis (MST) assay. We found that hydrophobic residues such as Phe23 are indispensable for M2-1 to recognize the Adenine (A) base of RNA. We also captured spontaneous binding of RNA (SH7) to the M2-1 protein in all-atom simulations using a robust Gaussian accelerated molecular dynamics (GaMD) method. Both the experiments and simulations revealed that two separate domains of M2-1 interact with RNA, suggesting that the recognition of RNA by the zinc-binding domain (ZBD) and binding of RNA by the core domain (CD) are independent of each other. Collectively, our results provided a structural basis for RNA recognition by HRSV M2-1. 

View Proposal 309

Author

Bo Liang, SOM, Emory University Atlanta, GA 

Additional Author(s)

Yunrong Gao, SOM, Emory University Atlanta, GA 
Dongdong Cao, SOM, Emory University Atlanta, GA 
Shristi Pawnikar, University of Kansas Lawrence, KS 
Yinglong Miao, University of Kansas Lawrence, KS 

Broad HIV neutralization by a vaccine-induced cow antibody

Potent broadly neutralizing antibodies to HIV have been very challenging to elicit by vaccination in wild-type animals. Using x-ray crystallography, cryo-electron microscopy and site-directed mutagenesis, we have analyzed the mode of binding of a potent bnAb (NC-Cow1) elicited in cows by immunization with the HIV gp140 Envelope trimer BG505 SOSIP.664. The exceptionally long (60 residues) third complementarity determining region of the NC-Cow1 heavy chain forms a knob-shaped mini-domain on an extended stalk that navigates through the dense glycan shield on the HIV envelope trimer to target a small footprint at the gp120 CD4 receptor binding site with no contact of the other CDRs to the rest of the Env trimer. These findings illustrate how an unusual vaccine-induced cow bnAb to HIV Env can neutralize with high potency and breadth. 

View Proposal 305

Author

Robyn Stanfield La Jolla, CA 

Correction of the allosteric site of E. coli D-3-phosphoglycerate dehydrogenase

D-3-phosphoglycerate dehydrogenase(PGDH), an enzyme in the serine biosynthesis pathway, is known as an example of "Vmax" allosteric regulation and as the archetype structure for the ACT conserved motif. The first structure of E. coli PGDH, published in 1995 by Schuller, et al., presented a tetramer with all of its allosteric sites filled with the inhibitor serine. With better data and new structures, we show here that the location of the serine was correct but its orientation was not. This error in the original structure was propagated to several other published structures. In 2005 a tetragonal structure was published by Thompson, et al. and touted as the uninhibited "active form" of the enzyme. We have duplicated this form with fresh data and structures, and have examined the deposited structure, and state here that this form consistently has one of the four serine sites in the tetramer occupied, which kinetic studies indicate should result in ~50% inhibition. 

View Proposal 306

Author

David Schuller, MacCHESS, Cornell Univ Ithaca, NY 

Additional Author

Tiit Lukk, Tallinn University of Technology Tallinn

High-resolution crystal structures of recombinant wild type and selenomethione labeled bovine trypsin (S195A) mutant reveals no electron density for three surface loops that includes the C191 - C220 disulfide.

Crystals of bovine trypsin (UniProt ID P00760) is used by SER-CAT as a quality control standard for determining beamline optimization and performance. However, a selenomethionine labeled standard for MAD/SAD energy optimization and other studies was also needed. To provide this MAD/SAD standard a S195A mutant of bovine trypsin with selenomethione labeling was expressed, purified and crystalized. Crystals of the recombinant protein had a similar habit and diffraction quality compared to native crystals. An 18 sec data set to 1.5 Å resolution was collected on beamline 22ID at the selenium absorption edge using a Rayonix MX300HS 10 Hz CCD detector. The data set (99.1% complete) consisting of 180 one-degree images each exposed for 0.1 seconds was collected. Following SER-CAT protocols the data were auto processed using cmdKylin. Phases were then generated using phenix.autosol and phenix.autobuild placed 190 of the 223 amino acids giving R and Rfree values of 0.2122 and 0.2332, respectively. Refinement (3 rounds) of the auobuilt model was carried out using phenix.refine and converged to give R and Rfree values of 0.1994 and 0.2187, respectively with good stereochemistry. However, inspection of the refined model (COOT) showed that the electron density of three surface loops (totaling 27 residues) was missing with the loss of the C191 - C220 disulfide. The presentation will provide details of the production of selenomethione labeled protein, its crystallization, data collection and structure solution. It will also explore possible causes for the missing loop density. Work supported in part by funds from the SER-CAT Member Institutions, the University of Georgia Foundation, and the National Institutes of Health (S10 RR25528 & 1S10 RR028976). 

View Proposal 432

Author

John Rose, SER-CAT/University of Georgia Athens, GA 

Additional Author(s)

Dayong Zhou, University of Georgia
Zheng-qing "Albert" Fu, SER-CAT/University of Georgia Argonne, IL 
Zhongmin Jin, SER-CAT/University of Georgia Argonne, IL 
John Gonczy, SER-CAT/University of Georgia Argonne, IL 
John Chrzas, SER-CAT/University of Georgia Argonne, IL 
Palani Kandavelu, SER-CAT/Department of Biochemistry & Molecular Biology Athens, GA 
Rodrick Salazar, SER-CAT/Department of Biochemisrtry and Molecular Biology Athens, GA 
Maaz Gul, Department of Biochemistry & Molecular Biology Athens, GA 
Lirong Chen, SER-CAT/University of Georgia Athens, GA 
Bi-Cheng Wang, University of Georgia
Unmesh Chinte, SER-CAT/University of Georgia Athens, GA