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      Structure and Interactions of the Human Programmed Cell Death 1 Receptor*


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          Background: The inhibitory leukocyte receptor PD-1 binds two ligands, PD-L1 and PD-L2.

          Results: Nuclear magnetic resonance analysis and rigorous binding and thermodynamic measurements reveal the structure of, and the mode of ligand recognition by, PD-1.

          Conclusion: PD-L1 and PD-L2 bind differently to PD-1 and much more weakly than expected.

          Significance: Potent inhibitory signaling can be initiated by weakly interacting receptors.


          PD-1, a receptor expressed by T cells, B cells, and monocytes, is a potent regulator of immune responses and a promising therapeutic target. The structure and interactions of human PD-1 are, however, incompletely characterized. We present the solution nuclear magnetic resonance (NMR)-based structure of the human PD-1 extracellular region and detailed analyses of its interactions with its ligands, PD-L1 and PD-L2. PD-1 has typical immunoglobulin superfamily topology but differs at the edge of the GFCC′ sheet, which is flexible and completely lacks a C″ strand. Changes in PD-1 backbone NMR signals induced by ligand binding suggest that, whereas binding is centered on the GFCC′ sheet, PD-1 is engaged by its two ligands differently and in ways incompletely explained by crystal structures of mouse PD-1·ligand complexes. The affinities of these interactions and that of PD-L1 with the costimulatory protein B7-1, measured using surface plasmon resonance, are significantly weaker than expected. The 3–4-fold greater affinity of PD-L2 versus PD-L1 for human PD-1 is principally due to the 3-fold smaller dissociation rate for PD-L2 binding. Isothermal titration calorimetry revealed that the PD-1/PD-L1 interaction is entropically driven, whereas PD-1/PD-L2 binding has a large enthalpic component. Mathematical simulations based on the biophysical data and quantitative expression data suggest an unexpectedly limited contribution of PD-L2 to PD-1 ligation during interactions of activated T cells with antigen-presenting cells. These findings provide a rigorous structural and biophysical framework for interpreting the important functions of PD-1 and reveal that potent inhibitory signaling can be initiated by weakly interacting receptors.

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          Most cited references36

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          MOLMOL: a program for display and analysis of macromolecular structures.

          MOLMOL is a molecular graphics program for display, analysis, and manipulation of three-dimensional structures of biological macromolecules, with special emphasis on nuclear magnetic resonance (NMR) solution structures of proteins and nucleic acids. MOLMOL has a graphical user interface with menus, dialog boxes, and on-line help. The display possibilities include conventional presentation, as well as novel schematic drawings, with the option of combining different presentations in one view of a molecule. Covalent molecular structures can be modified by addition or removal of individual atoms and bonds, and three-dimensional structures can be manipulated by interactive rotation about individual bonds. Special efforts were made to allow for appropriate display and analysis of the sets of typically 20-40 conformers that are conventionally used to represent the result of an NMR structure determination, using functions for superimposing sets of conformers, calculation of root mean square distance (RMSD) values, identification of hydrogen bonds, checking and displaying violations of NMR constraints, and identification and listing of short distances between pairs of hydrogen atoms.
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            Protein NMR structure determination with automated NOE assignment using the new software CANDID and the torsion angle dynamics algorithm DYANA.

            Combined automated NOE assignment and structure determination module (CANDID) is a new software for efficient NMR structure determination of proteins by automated assignment of the NOESY spectra. CANDID uses an iterative approach with multiple cycles of NOE cross-peak assignment and protein structure calculation using the fast DYANA torsion angle dynamics algorithm, so that the result from each CANDID cycle consists of exhaustive, possibly ambiguous NOE cross-peak assignments in all available spectra and a three-dimensional protein structure represented by a bundle of conformers. The input for the first CANDID cycle consists of the amino acid sequence, the chemical shift list from the sequence-specific resonance assignment, and listings of the cross-peak positions and volumes in one or several two, three or four-dimensional NOESY spectra. The input for the second and subsequent CANDID cycles contains the three-dimensional protein structure from the previous cycle, in addition to the complete input used for the first cycle. CANDID includes two new elements that make it robust with respect to the presence of artifacts in the input data, i.e. network-anchoring and constraint-combination, which have a key role in de novo protein structure determinations for the successful generation of the correct polypeptide fold by the first CANDID cycle. Network-anchoring makes use of the fact that any network of correct NOE cross-peak assignments forms a self-consistent set; the initial, chemical shift-based assignments for each individual NOE cross-peak are therefore weighted by the extent to which they can be embedded into the network formed by all other NOE cross-peak assignments. Constraint-combination reduces the deleterious impact of artifact NOE upper distance constraints in the input for a protein structure calculation by combining the assignments for two or several peaks into a single upper limit distance constraint, which lowers the probability that the presence of an artifact peak will influence the outcome of the structure calculation. CANDID test calculations were performed with NMR data sets of four proteins for which high-quality structures had previously been solved by interactive protocols, and they yielded comparable results to these reference structure determinations with regard to both the residual constraint violations, and the precision and accuracy of the atomic coordinates. The CANDID approach has further been validated by de novo NMR structure determinations of four additional proteins. The experience gained in these calculations shows that once nearly complete sequence-specific resonance assignments are available, the automated CANDID approach results in greatly enhanced efficiency of the NOESY spectral analysis. The fact that the correct fold is obtained in cycle 1 of a de novo structure calculation is the single most important advance achieved with CANDID, when compared with previously proposed automated NOESY assignment methods that do not use network-anchoring and constraint-combination.
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              Rapid measurement of binding constants and heats of binding using a new titration calorimeter.

              A new titration calorimeter is described and results are presented for the binding of cytidine 2'-monophosphate (2'CMP) to the active site of ribonuclease A. The instrument characteristics include very high sensitivity, rapid calorimetric response, and fast thermal equilibration. Convenient software is available for instrument operation, data collection, data reduction, and deconvolution to obtain least-squares estimates of binding parameters n, delta H degree, delta S degree, and the binding constant K. Sample through-put for the instrument is high, and under favorable conditions binding constants as large as 10(8) M-1 can be measured. The bovine ribonuclease A (RNase)/2'CMP system was studied over a 50-fold range of RNase concentration and at two different temperatures. The binding constants were in the 10(5) to 10(6) M-1 range, depending on conditions, and heats of binding ca. -15,000 cal/mol. Repeat determinations suggested errors of only a few percent in n, delta H degree, and K values over the most favorable concentration range.

                Author and article information

                J Biol Chem
                J. Biol. Chem
                The Journal of Biological Chemistry
                American Society for Biochemistry and Molecular Biology (9650 Rockville Pike, Bethesda, MD 20814, U.S.A. )
                26 April 2013
                15 February 2013
                15 February 2013
                : 288
                : 17
                : 11771-11785
                From the []Radcliffe Department of Medicine and
                [§ ]Medical Research Council Human Immunology Unit, University of Oxford, John Radcliffe Hospital, Headington, Oxford OX3 9DU, United Kingdom,
                the []Department of Biochemistry, University of Leicester, Leicester LE1 9HN, United Kingdom,
                the []Institute of Organic Chemistry and Biochemistry, Flemingovo Namesti 2, 166 10 Prague 6, Czech Republic,
                the [** ]Department of Biochemistry and Molecular Biology, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030,
                [‡‡ ]UCB Pharma, Slough SL1 4EN, United Kingdom,
                the [§§ ]Systems Biology Research Centre, School of Life Sciences, University of Skövde, Box 408, Skövde, Sweden, and
                the [¶¶ ]Division of Structural Biology, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Kumamoto 862 0973, Japan
                Author notes
                [2 ] To whom correspondence may be addressed. E-mail: mdc12@ 123456le.ac.uk .
                [3 ] To whom correspondence may be addressed. E-mail: simon.davis@ 123456imm.ox.ac.uk .

                Both authors contributed equally to this work.

                © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

                Author's Choice—Final version full access.

                Creative Commons Attribution Unported License applies to Author Choice Articles

                : 21 December 2012
                : 11 February 2013

                cell surface protein,nuclear magnetic resonance,receptors,signaling,surface plasmon resonance (spr),affinity,complex formation,thermodynamics


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