1. Academic Validation
  2. Crystal structure of the m4-1BB/4-1BBL complex reveals an unusual dimeric ligand that undergoes structural changes upon 4-1BB receptor binding

Crystal structure of the m4-1BB/4-1BBL complex reveals an unusual dimeric ligand that undergoes structural changes upon 4-1BB receptor binding

  • J Biol Chem. 2019 Feb 8;294(6):1831-1845. doi: 10.1074/jbc.RA118.006297.
Aruna Bitra 1 Tzanko Doukov 2 Giuseppe Destito 3 Michael Croft 1 4 Dirk M Zajonc 5 6
Affiliations

Affiliations

  • 1 From the Division of Immune Regulation, La Jolla Institute for Immunology (LJI), La Jolla, California 92037.
  • 2 the Stanford Synchrotron Radiation Lightsource, SLAC, Menlo Park, California 94025.
  • 3 Kirin Kyowa Hakko Pharmaceutical Research, La Jolla, California 92037.
  • 4 the Department of Medicine, University of California San Diego, La Jolla, California 92037, and.
  • 5 From the Division of Immune Regulation, La Jolla Institute for Immunology (LJI), La Jolla, California 92037, dzajonc@lji.org.
  • 6 the Department of Internal Medicine, Faculty of Medicine and Health Sciences, Ghent University, 9000 Ghent, Belgium.
Abstract

The interaction between the receptor 4-1BB and its ligand 4-1BBL provides co-stimulatory signals for T-cell activation and proliferation. However, differences in the mouse and human molecules might result in differential engagement of this pathway. Here, we report the crystal structure of mouse 4-1BBL and of the mouse 4-1BB/4-1BBL complex, which together provided insights into the molecular mechanism by which m4-1BBL and its cognate receptor recognize each other. Unlike all human or mouse tumor necrosis factor ligands that form noncovalent and mostly trimeric assemblies, the m4-1BBL structure formed a disulfide-linked dimeric assembly. The structure disclosed that certain differences in the amino acid composition along the intramolecular interface, together with two specific residues (Cys-246 and Ser-256) present exclusively in m4-1BBL, are responsible for this unique dimerization. Unexpectedly, upon m4-1BB binding, m4-1BBL undergoes structural changes within each protomer; moreover, the individual m4-1BBL protomers rotate relative to each other, yielding a dimerization interface with more inter-subunit interactions. We also observed that in the m4-1BB/4-1BBL complex, each receptor monomer binds exclusively to a single ligand subunit with contributions of cysteine-rich domain 1 (CRD1), CRD2, and CRD3. Furthermore, structure-guided mutagenesis of the binding interface revealed that novel binding interactions with the GH loop, rather than the DE loop, are energetically critical and define the m4-1BB receptor selectivity for m4-1BBL. A comparison with the human 4-1BB/4-1BBL complex highlighted several differences between the ligand- and receptor-binding interfaces, providing an explanation for the absence of inter-species cross-reactivity between human and mouse 4-1BB and 4-1BBL molecules.

Keywords

4-1BB; 4-1BB ligand; CD137; CD137L; TNFRSF9; X-ray crystallography; cell surface receptor; immune cell; protein structure; protein–protein interaction; tumor necrosis factor (TNF).

Figures