To address the mechanism of neutrophil activation, we modeled Fg and intact M1 in place of FgD and M1BC1
(, Supplementary Movie 1
), respectively. Importantly, because Fg is a dimer (of αβγ heterotrimers), a Fg molecule has two M1-binding sites as opposed to the single site in FgD. From this modeling emerged a non-clashing M1-Fg network with Fg acting as struts and M1 acting as joints. The two M1 molecules that bound an individual Fg were tilted with respect to each other due to the inherent flexibility of Fg21
. This tilt gave the network three-dimensional character and meant that the network incorporated M1 molecules pointing in opposite directions. The variation in M1 directionality suggests that the network is formed by free M1 released from the bacterial surface by neutrophil proteases6
, as opposed to M1 anchored unidirectionally by its C-terminus to the bacterial cell wall22
. Consistent with this notion, the greater proportion of M1 in samples from STSS patients occurs as free released protein7
The structure of the M1-Fg network suggested a mechanism for neutrophil activation. Prior work had shown that antibody crosslinking of β2
integrin had the same effect on neutrophil activation as the M1-Fg complex6,9,23
, indicating that β2
integrin clustering and avidity are involved in signaling by M1-Fg. Based on these data, we surmised that the Fg density induced by the M1-Fg network was likely to be a critical factor for neutrophil activation. To test this model, we compared HBP release by neutrophils stimulated by various M1 deletion constructs. M1 in which either the upstream or downstream B-repeat was deleted, ΔB1 and ΔB2, respectively, retained Fg binding due to the continued presence of one of the B-repeats (). However, as the modeling predicted, ΔB1 and ΔB2 formed fibers ( and Supplementary Fig. 5a
) rather than the networks formed by wild-type M1 (). Despite being able to bind Fg, neither ΔB1 nor ΔB2 triggered release of HBP from neutrophils, which was in sharp contrast to wild-type M1 (). This result indicates that the M1-Fg network rather than Fg-binding itself is required for neutrophil activation. We also demonstrated that the addition of FgD, which is unable to support network formation because it has only one M1-binding site, blocks M1-mediated neutrophil activation (). Excess FgD was necessary in this experiment as binding of M1 to FgD is weaker than it is to Fg24
, the difference being explained by the high avidity between M1 and Fg as compared to the weaker affinity between M1 and FgD.
Fg binding and neutrophil activation
As expected, deletion of both B-repeats (ΔB1B2) resulted in no networks, no fibers, and no induction of HBP release ( and Supplementary Fig. 5b
). However, ΔB1B2 retained a low level of Fg binding (). Based on this and other evidence, we uncovered a cryptic Fg-binding site in the A-region. A molecular replacement solution of a low-resolution crystal (7.5 Å resolution limit) of the M1 A-region bound to FgD (M1A
-FgD) confirmed the existence of this site. This solution revealed two molecules of FgD oriented 180° to one another and arranged perpendicularly to the A-region, similar to the binding mode observed for each of the B-repeats (Supplementary Fig. 6
). Although the low resolution limited our abilities to discern A-region residues, it was apparent that the same FgD residues bound by the B-repeats were bound by the A-region. This suggested A-region residues 106-119 to be the likely Fg-binding site (Supplementary Fig. 7
), as this region has some sequence similarity to the B-repeats, including a Tyr capable of forming a π-cation bond to FgD β169; tyrosines are otherwise rare in the M1 sequence. In line with observations for the B-repeats, the A-region site would also require a ~51.4° rotation in helical register from the conformation observed in M1AB
) to bind Fg. Deletion of this putative Fg-binding site in the A-region along with both B-repeats completely abrogated Fg binding (, Δ98ΔB1B2). While we found that the A-region site was not required for network formation or for HBP release (, Δ98; Supplementary Fig. 5c
), the possibility that this cryptic site has other functions related to Fg binding (e.g., evasion of phagocytosis) merits future exploration.
Lastly, we asked whether the density of the M1-Fg network was consequential. To address this, we deleted the downstream B-repeat (B2) and inserted it at the C-terminus of M1, thereby increasing the spacing between the two B-repeats (). Modeling predicted that a sparser network should be formed by this construct, called B2C. B2C bound FgD and formed networks, but significantly, did not trigger release of HBP ( and ). We note that the resolution of the electron micrographs did not allow us to distinguish between M1-Fg and B2C-Fg networks, but our modeling strongly suggests that the difference in network density accounted for the lack of neutrophil activation.