Remarkably low affinity of CD4/peptide-major histocompatibility complex class II protein interactions.
The αβ T-cell coreceptor CD4 enhances immune responses more than 1 million-fold in some assays, and yet the affinity of CD4 for its ligand, peptide-major histocompatibility class II (pMHC II) on antigen-presenting cells, is so weak that it was previously unquantifiable. Here, we report that a soluble form of CD4 failed to bind detectably to pMHC II in surface plasmon resonance-based assays, establishing a new upper limit for the solution affinity at 2.5 mM. However, when presented multivalently on magnetic beads, soluble CD4 bound pMHC II-expressing B cells, confirming that it is active and allowing mapping of the native coreceptor binding site on pMHC II. Whereas binding was undetectable in solution, the affinity of the CD4/pMHC II interaction could be measured in 2D using CD4- and adhesion molecule-functionalized, supported lipid bilayers, yielding a 2D Kd of ∼5,000 molecules/μm(2) This value is two to three orders of magnitude higher than previously measured 2D Kd values for interacting leukocyte surface proteins. Calculations indicated, however, that CD4/pMHC II binding would increase rates of T-cell receptor (TCR) complex phosphorylation by threefold via the recruitment of Lck, with only a small, 2-20% increase in the effective affinity of the TCR for pMHC II. The affinity of CD4/pMHC II therefore seems to be set at a value that increases T-cell sensitivity by enhancing phosphorylation, without compromising ligand discrimination.
Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, United Kingdom; Department of Chemistry, Lund University, SE-22100 Lund, Sweden;
Peter
P
Jönsson
MRC Human Immunology Unit, University of Oxford, Oxford OX3 9DS, United Kingdom; Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, United Kingdom; Nuffield Department of Obstetrics and Gynaecology, University of Oxford, Oxford OX3 9DU, United Kingdom;
Jennifer H
JH
Southcombe
MRC Human Immunology Unit, University of Oxford, Oxford OX3 9DS, United Kingdom; Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, United Kingdom;
Ana Mafalda
AM
Santos
MRC Human Immunology Unit, University of Oxford, Oxford OX3 9DS, United Kingdom; Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, United Kingdom;
Jiandong
J
Huo
MRC Human Immunology Unit, University of Oxford, Oxford OX3 9DS, United Kingdom; Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, United Kingdom;
Ricardo A
RA
Fernandes
Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, United Kingdom;
James
J
McColl
Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom;
Melissa
M
Lever
MRC Human Immunology Unit, University of Oxford, Oxford OX3 9DS, United Kingdom; Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, United Kingdom;
Edward J
EJ
Evans
MRC Human Immunology Unit, University of Oxford, Oxford OX3 9DS, United Kingdom; Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, United Kingdom;
Alexander
A
Hudson
MRC Human Immunology Unit, University of Oxford, Oxford OX3 9DS, United Kingdom; Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, United Kingdom;
Veronica T
VT
Chang
MRC Human Immunology Unit, University of Oxford, Oxford OX3 9DS, United Kingdom;
Tomáš
T
Hanke
Department of Infection and Immunity, School of Medicine, Cardiff University, Cardiff CF14 4XN, United Kingdom;
Andrew
A
Godkin
Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, United Kingdom;
Paul D
PD
Dunne
Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, United Kingdom;
Mathew H
MH
Horrocks
Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, United Kingdom;
Matthieu
M
Palayret
MRC Human Immunology Unit, University of Oxford, Oxford OX3 9DS, United Kingdom;
Gavin R
GR
Screaton
Infection and Immunity Program and Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, VIC 3800, Australia; Australian Research Council Centre of Excellence in Advanced Molecular Imaging, Monash University, Clayton, VIC 3800, Australia;
Jan
J
Petersen
Department of Infection and Immunity, School of Medicine, Cardiff University, Cardiff CF14 4XN, United Kingdom; Infection and Immunity Program and Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, VIC 3800, Australia; Australian Research Council Centre of Excellence in Advanced Molecular Imaging, Monash University, Clayton, VIC 3800, Australia;
Jamie
J
Rossjohn
MRC Human Immunology Unit, University of Oxford, Oxford OX3 9DS, United Kingdom; Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, United Kingdom; Nuffield Department of Clinical Neurosciences, Division of Clinical Neurology, University of Oxford, Oxford OX3 9DS, United Kingdom.
Lars
L
Fugger
Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom;
Omer
O
Dushek
MRC Human Immunology Unit, University of Oxford, Oxford OX3 9DS, United Kingdom; x.xu@imperial.ac.uk simon.davis@imm.ox.ac.uk dk10012@cam.ac.uk.
Xiao-Ning
XN
Xu
MRC Human Immunology Unit, University of Oxford, Oxford OX3 9DS, United Kingdom; Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, United Kingdom; x.xu@imperial.ac.uk simon.davis@imm.ox.ac.uk dk10012@cam.ac.uk.
Simon J
SJ
Davis
Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, United Kingdom; x.xu@imperial.ac.uk simon.davis@imm.ox.ac.uk dk10012@cam.ac.uk.
David
D
Klenerman
D001665:Binding Sites; D015704:CD4 Antigens / Q000737:chemistry* / Q000378:metabolism; D057809:HEK293 Cells; D059871:HLA-A24 Antigen / Q000737:chemistry* / Q000378:metabolism; D059811:HLA-DRB1 Chains / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D058619:Maltose-Binding Proteins / Q000737:chemistry; D008958:Models, Molecular; D010766:Phosphorylation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D011499:Protein Processing, Post-Translational; D055550:Protein Stability; D020349:Surface Plasmon Resonance
D015704:CD4 Antigens; C438506:HLA-A*24:02 antigen; D059871:HLA-A24 Antigen; D059811:HLA-DRB1 Chains; C489631:HLA-DRB1*15:01 antigen; D058619:Maltose-Binding Proteins
T-cell activation; TCR phosphorylation; adhesion; binding equilibrium and kinetics; protein interactions