Structural Determinants of Herpesvirus Entry Mediator Recognition by Murine B and T Lymphocyte Attenuator

Christopher A Nelson,Daved H Fremont,Kenneth M Murphy,John R Sedy,Carl F Ware,Marcel D Fremont,Paula S Norris

doi:10.4049/jimmunol.180.2.940

Christopher A Nelson, Daved H Fremont + Show 5 more

Open Access

https://doi.org/10.4049/jimmunol.180.2.940

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Abstract

The B and T lymphocyte attenuator (BTLA) appears to act as a negative regulator of T cell activation and growth. BTLA specifically interacts with herpesvirus entry mediator (HVEM), a member of the TNFR family. Herein, we have undertaken surface plasmon resonance studies to quantitatively assess BTLA and HVEM ectodomain interactions. We find that soluble BALB/cJ BTLA engages HVEM with an equilibrium affinity of 0.97+/-0.19 microM while the C57BL/6 BTLA binds slightly better with an equilibrium affinity of 0.42+/-0.06 microM. Despite its lower affinity for HVEM, the kinetic half-life of BALB/cJ BTLA complexes are twice as long as observed for C57BL/6 BTLA (4 vs 2 s). To further explore these interactions, we solved the crystal structure of a murine BTLA (BALB/cJ) ectodomain at 1.8-A resolution, revealing a beta sandwich fold with strong similarity to I-set members of the Ig superfamily. Using a structure-based mutagenesis strategy, we then examined the individual contributions of 26 BTLA surface-exposed residues toward HVEM binding. Four single-site substitutions were identified that decrease HVEM binding below detectable levels and two that decrease binding by more than half. All six of these cluster at the edge of the beta sandwich in a membrane distal patch formed primarily from the A and G strands. This patch falls within the contacting surface recently revealed in the crystal structure of the human BTLA-HVEM cocomplex. The critical binding residues identified here are highly conserved across species, suggesting that BTLA employs a conserved binding mode for HVEM recognition.

Highlights

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We find that soluble BALB/cJ B and T lymphocyte attenuator (BTLA) engages herpesvirus entry mediator (HVEM) with an equilibrium affinity of 0.97 ؎ 0.19 ␮M while the C57BL/6 BTLA binds slightly better with an equilibrium affinity of 0.42 ؎ 0.06 ␮M
The sequences currently available indicate that polymorphism of murine HVEM is very limited, the sequence of BALB/cJ HVEM has not been reported and it is not known whether the BALB/cJ and C57BL/6 HVEM proteins are identical at the BTLA interaction site

Summary

Abbreviations used in this paper

BTLA, B and T lymphocyte attenuator; HVEM, herpesvirus entry mediator; CRD, cysteine-rich domain; PD-1, programmed death receptor 1; ESMS, electrospray mass spectrometry; SPR, surface plasmon resonance; GVHD, graft-versus-host disease. BTLA recognizes, as its principal binding target, an extended ␤-strand located in the membrane-distal region of the HVEM CRD1 domain, HVEM ␤-strand residues 35–39 (TVCEP) These residues form an intramolecular anti-parallel ␤-sheet with residues 122–128 (NLIESHS) of human BTLA. When alaninescanning mutagenesis was used to evaluate the binding contributions of 15 individual HVEM side chains, the three most disruptive substitutions were residues involved in the formation of the ␤-strand, confirming the intramolecular ␤-sheet as a central feature of the binding interface. These results suggest BTLA uses a unique binding surface, distinct from that used by coinhibitory receptors of the related CD28 family. Our results confirm that murine and human BTLA engage HVEM using a conserved binding motif and represent the first functional characterization of individual BTLA residues involved in HVEM binding

Materials and Methods

Results

Discussion