CaltechAUTHORS
  A Caltech Library Service

High-resolution crystal structure of human asparagine synthetase enables analysis of inhibitor binding and selectivity

Zhu, Wen and Radadiya, Ashish and Bisson, Claudine and Wenzel, Sabine and Nordin, Brian E. and Martínez-Márquez, Francisco and Imasaki, Tsuyoshi and Sedelnikova, Svetlana E. and Coricello, Adriana and Baumann, Patrick and Berry, Alexandria H. and Nomanbhoy, Tyzoon K. and Kozarich, John W. and Jin, Yi and Rice, David W. and Takagi, Yuichiro and Richards, Nigel G. J. (2019) High-resolution crystal structure of human asparagine synthetase enables analysis of inhibitor binding and selectivity. Communications Biology, 2 . Art. No. 345. ISSN 2399-3642. PMCID PMC6748925. https://resolver.caltech.edu/CaltechAUTHORS:20190930-133756450

[img] PDF - Published Version
Creative Commons Attribution.

2403Kb
[img] PDF - Supplemental Material
Creative Commons Attribution.

8Mb
[img] MS Word (Description of Additional Supplementary Files) - Supplemental Material
Creative Commons Attribution.

13Kb
[img] MS Excel (Supplementary Data 1) - Supplemental Material
Creative Commons Attribution.

54Kb
[img] MS Excel (Supplementary Data 2) - Supplemental Material
Creative Commons Attribution.

24Kb
[img] MS Word (Supplementary Data 3) - Supplemental Material
Creative Commons Attribution.

13Kb
[img] PDF (Reporting Summary) - Supplemental Material
Creative Commons Attribution.

188Kb

Use this Persistent URL to link to this item: https://resolver.caltech.edu/CaltechAUTHORS:20190930-133756450

Abstract

Expression of human asparagine synthetase (ASNS) promotes metastatic progression and tumor cell invasiveness in colorectal and breast cancer, presumably by altering cellular levels of L-asparagine. Human ASNS is therefore emerging as a bona fide drug target for cancer therapy. Here we show that a slow-onset, tight binding inhibitor, which exhibits nanomolar affinity for human ASNS in vitro, exhibits excellent selectivity at 10 μM concentration in HCT-116 cell lysates with almost no off-target binding. The high-resolution (1.85 Å) crystal structure of human ASNS has enabled us to identify a cluster of negatively charged side chains in the synthetase domain that plays a key role in inhibitor binding. Comparing this structure with those of evolutionarily related AMP-forming enzymes provides insights into intermolecular interactions that give rise to the observed binding selectivity. Our findings demonstrate the feasibility of developing second generation human ASNS inhibitors as lead compounds for the discovery of drugs against metastasis.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1038/s42003-019-0587-zDOIArticle
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6748925/PubMed CentralArticle
ORCID:
AuthorORCID
Zhu, Wen0000-0003-3190-0071
Radadiya, Ashish0000-0001-7348-1755
Wenzel, Sabine0000-0001-8436-6226
Imasaki, Tsuyoshi0000-0001-5462-1820
Coricello, Adriana0000-0002-3306-9261
Baumann, Patrick0000-0001-9484-7402
Kozarich, John W.0000-0002-9652-2289
Jin, Yi0000-0002-6927-4371
Rice, David W.0000-0002-7811-0539
Takagi, Yuichiro0000-0002-0988-6798
Richards, Nigel G. J.0000-0002-0375-0881
Additional Information:© The Author(s) 2019. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Received: 6 September 2018. Accepted: 21 August 2019. Published 17 September 2019. We are grateful to Dr. H. Ikeuchi and Professor J. Hiratake (Kyoto) for generously providing samples of the human ASNS inhibitor 1 used in the functional proteomics experiments. We thank the Diamond Light Source for beam time allocation and beam line staff for assistance with data collection. Mass spectrometric characterizations of human ASNS and the DON-modified variant were performed at the University of Sheffield Biological Mass Spectrometry Facility. Author Contributions: W.Z., C.B., and S.E.S.: performed purification and crystallization of human ASNS. W.Z. and C.B.: performed crystallographic studies and obtained the refined X-ray crystal structure of human ASNS. W.Z.: performed Dali structural similarity studies, HCT-116 transcriptome analysis and kinetic characterization of the ASD-linked ASNS variants. T.I., S.W., F.M.M. and Y.T.: designed the baculovirus constructs and expressed the recombinant forms of human ASNS and human ASS1. A.R.: performed the inhibitor stability measurements, computational modeling studies and free energy calculations. B.E.N. and T.K.N.: devised and performed the chemoproteomic profiling experiments. Y.J., P.B., A.C. and A.H.B.: performed kinetic/inhibition assays on WT human ASNS and site-specific variants. W.Z., J.W.K., D.W.R., Y.T. and N.G.J.R.: designed the study. W.Z., Y.T. and N.G.J.R.: wrote the manuscript with contributions from all other authors. Data availability: Atomic coordinates and structure factors for recombinant, DON-modified human ASNS have been deposited in the Protein Data Bank with accession number 6GQ3. Coordinates for the computational models of the 1a/MgPPi/ASNS, 1b/MgPPi/ASNS and β–aspartyl-AMP/MgPPi/ASNS complexes, MD simulation trajectories and I/O files for the free energy calculations, and raw data for protein purification and kinetic assays are available from Professor Nigel Richards (RichardsN14@cardiff.ac.uk) on request. Requests for plasmids and other reagents needed to obtain the ASNS variants used in this study should be sent to Professor Yuichiro Takagi (ytakagi@iu.edu). Raw data for the chemoproteomic profiling experiments can be obtained by contacting Dr. Tyzoon Nomanbhoy (tyzoonn@ACTIVX.com). The authors declare no competing interests.
Funders:
Funding AgencyGrant Number
Biotechnology and Biological Sciences Research Council (BBSRC)BB/I003703/1
NIHGM111695
PubMed Central ID:PMC6748925
Record Number:CaltechAUTHORS:20190930-133756450
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20190930-133756450
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:98954
Collection:CaltechAUTHORS
Deposited By: George Porter
Deposited On:30 Sep 2019 22:30
Last Modified:03 Oct 2019 21:45

Repository Staff Only: item control page