The role of disulfide bonds in the assembly and function of MD-2

Abstract

MD-2 is a secreted glycoprotein that binds to the extracellular domain of Toll-like receptor 4 (TLR4) and is required for the activation of TLR4 by lipopolysaccharide (LPS). The protein contains seven Cys residues and consists of a heterogeneous collection of disulfide-linked oligomers. To investigate the role of sulfhydryls in MD-2 structure and function, we created 17 single and multiple Cys substitution mutants. All of the MD-2 mutant proteins, including one totally lacking Cys residues, were secreted and stable. SDSPAGE analyses indicated that most Cys residues could participate in oligomer formation and that no single Cys residue was required for oligomerization. Of the single Cys substitutions, only C95S and C105S failed to confer LPS responsiveness on TLR4 when mutant and TLR4 were cotransfected into cells expressing an NF-kappaB reporter plasmid. Surprisingly, substitution of both C95 and C105 partially restored activity. Structural analyses revealed that C95 and C105 formed an intrachain disulfide bond, whereas C95 by itself produced an inactive dimer. In contrast to the cotransfection experiments, only WT MD-2 conferred responsiveness to LPS when secreted proteins were added directly to TLR4 reporter cells. Our data are consistent with a model in which most, possibly all sulfhydryls lie on the surface of a stable MD-2 core structure where they form both intra- and interchain disulfide bridges. These disulfide bonds produce a heterogeneous array of oligomers, including some species that can form an active complex with TLR4.

Figure 1

Effects of Cys substitutions on MD-2 activity. HEK293 cells were cotransfected with reporter vectors, hTLR4, and empty vector (V), WT, or mutant MD-2. Transfectants were treated with (filled bars) or without (open bars) LPS, and NF-κB activities were determined and normalized to the activity of WT MD-2 in the presence of LPS. Each bar represents the mean and SEM of at least nine replicated points from three or more independent experiments. Residues that were mutated from Cys (C) to Ser (S) are indicated in the table below the graph, and mutant designations are specified on the x axis.

Figure 2

Cys mutants are inactive in add-back experiments. TLR4 reporter cells were incubated with LPS and serial dilutions of 20-fold concentrated supernatants containing WT MD-2 or mutants 4, 6, 9, or 14. Concentrated supernatants from untransfected HEK293T cells did not confer LPS responsiveness (not shown).

Figure 3

Differential effects of single Cys substitutions on MD-2 oligomerization. WT or mutant MD-2 proteins were immunoprecipitated from transiently transfected HEK293T culture supernatants and analyzed by SDS/PAGE under nonreducing (NR and PR) or reducing (R) conditions. Samples designated PR were reduced and alkylated under nondenaturing conditions before analysis by SDS/PAGE. M, monomer; D, dimer; P, polymer. Mo refers to supernatant from mock-transfected HEK293T cells. In NR and PR panels, exposures were chosen to best show oligomer distributions. No MD-2 monomer was seen in WT or any single Cys mutant even at much higher exposure (not shown). In R, all samples were run at the same exposure and are the same samples used in add-back experiments (Fig. 6).

Figure 4

Polymer distributions of MD-2 mutants containing multiple Cys substitutions. MD-2 protein containing multiple Cys to Ser substitutions were treated as in Fig. 3.

Figure 5

Diagram of proposed MD-2 structures. (A) Mutant 10 is observed as a dimer, indicating that C95 is able to form an interchain disulfide. (B) The formation of an intrachain disulfide bond between C95 and C105 explains why mutant 11 is mainly monomeric. This intrachain disulfide prevents the formation of the C95-C95 interchain disulfide bridge observed in mutant 10. However, some dimer is observed and can be explained by the formation of either two C95-C105 or C95-C95 and C105-C105 interchain disulfide bonds. (C) Possible structures for dimeric and tetrameric WT MD-2. MD-2 dimers contain mixtures of inter- and intrachain disulfide bonds. The intrachain bonds from two dimers can alternatively form interchain bridges leading to tetramer formation. A similar process could generate the distribution of polymers seen in the WT lanes of Figs. 3 and 4.