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      An expanded allosteric network in PTP1B by multitemperature crystallography, fragment screening, and covalent tethering

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          Abstract

          Allostery is an inherent feature of proteins, but it remains challenging to reveal the mechanisms by which allosteric signals propagate. A clearer understanding of this intrinsic circuitry would afford new opportunities to modulate protein function. Here, we have identified allosteric sites in protein tyrosine phosphatase 1B (PTP1B) by combining multiple-temperature X-ray crystallography experiments and structure determination from hundreds of individual small-molecule fragment soaks. New modeling approaches reveal 'hidden' low-occupancy conformational states for protein and ligands. Our results converge on allosteric sites that are conformationally coupled to the active-site WPD loop and are hotspots for fragment binding. Targeting one of these sites with covalently tethered molecules or mutations allosterically inhibits enzyme activity. Overall, this work demonstrates how the ensemble nature of macromolecular structure, revealed here by multitemperature crystallography, can elucidate allosteric mechanisms and open new doors for long-range control of protein function.

          eLife digest

          Proteins perform many important jobs in each of the cells in our bodies, such as transporting other molecules and helping chemical reactions to occur. The part of the protein directly involved in these tasks is called the active site. Other areas of the protein can communicate with the active site to switch the protein on or off. This method of control is known as allostery.

          Switching proteins on and off could help us to develop treatments for certain diseases. For example, a protein called PTP1B reduces how well cells can respond to insulin. Switching this protein off could therefore help to treat diabetes. However, much like it’s hard to guess how a light switch is wired to a light bulb without seeing behind the walls, it is hard to predict which remote areas of a protein are ‘wired’ to the active site.

          Keedy, Hill et al. have now used two complementary methods to examine the structure of PTP1B and find new allosteric sites. The first method captured a series of X-ray images from crystallized molecules of the protein held at different temperatures. This revealed areas of PTP1B that can move like windshield wipers to communicate with each other. The second method soaked PTP1B crystals in trays with hundreds of drug-sized molecules and assessed which sites on the protein the molecules bound to. The molecules generally bound to just a few sites of the protein. Further tests on one of these sites showed that it can communicate with the active site to turn the protein on or off.

          Further work will be needed to develop drugs that could treat diabetes by binding to the newly identified allosteric sites in PTP1B. More generally, the methods developed by Keedy, Hill et al. could be used to study allostery in other important proteins.

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          Most cited references51

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          Linking crystallographic model and data quality.

          In macromolecular x-ray crystallography, refinement R values measure the agreement between observed and calculated data. Analogously, R(merge) values reporting on the agreement between multiple measurements of a given reflection are used to assess data quality. Here, we show that despite their widespread use, R(merge) values are poorly suited for determining the high-resolution limit and that current standard protocols discard much useful data. We introduce a statistic that estimates the correlation of an observed data set with the underlying (not measurable) true signal; this quantity, CC*, provides a single statistically valid guide for deciding which data are useful. CC* also can be used to assess model and data quality on the same scale, and this reveals when data quality is limiting model improvement.
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            The energy landscapes and motions of proteins

            Recent experiments, advances in theory, and analogies to other complex systems such as glasses and spin glasses yield insight into protein dynamics. The basis of the understanding is the observation that the energy landscape is complex: Proteins can assume a large number of nearly isoenergetic conformations (conformational substates). The concepts that emerge from studies of the conformational substates and the motions between them permit a quantitative discussion of one simple reaction, the binding of small ligands such as carbon monoxide to myoglobin.
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              Allosteric inhibition of SHP2 phosphatase inhibits cancers driven by receptor tyrosine kinases.

              The non-receptor protein tyrosine phosphatase SHP2, encoded by PTPN11, has an important role in signal transduction downstream of growth factor receptor signalling and was the first reported oncogenic tyrosine phosphatase. Activating mutations of SHP2 have been associated with developmental pathologies such as Noonan syndrome and are found in multiple cancer types, including leukaemia, lung and breast cancer and neuroblastoma. SHP2 is ubiquitously expressed and regulates cell survival and proliferation primarily through activation of the RAS–ERK signalling pathway. It is also a key mediator of the programmed cell death 1 (PD-1) and B- and T-lymphocyte attenuator (BTLA) immune checkpoint pathways. Reduction of SHP2 activity suppresses tumour cell growth and is a potential target of cancer therapy. Here we report the discovery of a highly potent (IC50 = 0.071 μM), selective and orally bioavailable small-molecule SHP2 inhibitor, SHP099, that stabilizes SHP2 in an auto-inhibited conformation. SHP099 concurrently binds to the interface of the N-terminal SH2, C-terminal SH2, and protein tyrosine phosphatase domains, thus inhibiting SHP2 activity through an allosteric mechanism. SHP099 suppresses RAS–ERK signalling to inhibit the proliferation of receptor-tyrosine-kinase-driven human cancer cells in vitro and is efficacious in mouse tumour xenograft models. Together, these data demonstrate that pharmacological inhibition of SHP2 is a valid therapeutic approach for the treatment of cancers.
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                Author and article information

                Contributors
                Role: Reviewing Editor
                Journal
                eLife
                Elife
                eLife
                eLife
                eLife Sciences Publications, Ltd
                2050-084X
                07 June 2018
                2018
                : 7
                : e36307
                Affiliations
                [1 ]deptDepartment of Bioengineering and Therapeutic Sciences University of California, San Francisco San FranciscoUnited States
                [2 ]deptDepartment of Pharmaceutical Chemistry University of California, San Francisco San FranciscoUnited States
                [3 ]deptCellular and Molecular Pharmacology University of California, San Francisco San FranciscoUnited States
                [4 ]Diamond Light Source DidcotUnited Kingdom
                [5 ]deptCrystal and Structural Chemistry Group, Bijvoet Center for Biomolecular Research Utrecht University UtrechtNetherlands
                [6 ]deptStructural Genomics Consortium University of Oxford OxfordUnited Kingdom
                [7 ]deptDepartment of Biochemistry University of Johannesburg JohannesburgSouth Africa
                [8]DE Shaw Research United States
                [9]DE Shaw Research United States
                Author notes
                [‡]

                Structural Biology Initiative, CUNY Advanced Science Research Center, New York, United States.

                [§]

                Department of Chemistry and Biochemistry, City College of New York, New York, United States.

                [#]

                Biochemistry and Chemistry PhD Programs, Graduate Center, City University of New York, New York, United States.

                [¶]

                Jnana Therapeutics, Cambridge, United States.

                *- equal contribution.

                [†]

                These authors contributed equally to this work.

                Author information
                http://orcid.org/0000-0002-9184-7586
                http://orcid.org/0000-0002-0935-8362
                https://orcid.org/0000-0001-6015-320X
                http://orcid.org/0000-0002-6693-8603
                http://orcid.org/0000-0003-0378-0017
                http://orcid.org/0000-0001-8267-5519
                http://orcid.org/0000-0002-5080-2859
                Article
                36307
                10.7554/eLife.36307
                6039181
                29877794
                1f8a4214-a6a6-4351-9df1-6eaca0198165
                © 2018, Keedy et al

                This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

                History
                : 01 March 2018
                : 04 June 2018
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/100002112, A.P. Giannini Foundation;
                Award ID: Postdoctoral Fellowship
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100005237, Helen Hay Whitney Foundation;
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000054, National Cancer Institute;
                Award ID: K99CA203002
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000054, National Cancer Institute;
                Award ID: F31 CA180378
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100010269, Wellcome Trust;
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000054, National Cancer Institute;
                Award ID: CA191018
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100005665, Kinship Foundation;
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000875, Pew Charitable Trusts;
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000008, David and Lucile Packard Foundation;
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000057, National Institute of General Medical Sciences;
                Award ID: GM110580
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000001, National Science Foundation;
                Award ID: STC-1231306
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100005595, University of California;
                Award ID: LFR-17-476732
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000057, National Institute of General Medical Sciences;
                Award ID: GM123159
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000057, National Institute of General Medical Sciences;
                Award ID: GM124169
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000057, National Institute of General Medical Sciences;
                Award ID: GM124149
                Award Recipient :
                The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
                Categories
                Research Article
                Structural Biology and Molecular Biophysics
                Custom metadata
                By measuring how multiple conformations shift in response to temperature, a new allosteric binding site is discovered for the phosphatase PTP1B.

                Life sciences
                allostery,protein dynamics,phosphatase,e. coli,human
                Life sciences
                allostery, protein dynamics, phosphatase, e. coli, human

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