Photo-Cross-Linking Mass Spectrometry and Integrative Modeling Enables Rapid Screening of Antigen Interactions Involving Bacterial Transferrin Receptors

Structure-based
approaches to the delineation of immunogens for
vaccine development have a throughput requirement that is difficult
to meet in practice with conventional methods of structure determination.
Here we present a strategy for rapid and accurate structure generation
in support of antigen engineering programs. The approach is developed
around the modeling of interactions between host transferrin (Tf)
and the bacterial vaccine target transferrin binding protein B (TbpB)
from Gram-negative pathogens such as Neisseria meningitidis. Using an approach based solely on cross-linking mass spectrometry
(XL-MS) data, monomeric structural models, and the Integrative Modeling
Platform (IMP), we demonstrate that converged representations of the
Tf:TbpB interactions can be returned that accurately reflect the binding
interface and the relative orientation of the monomeric units, with
the capacity to scale to the analysis of interactions from any number
of additional strains. We show that a key element to accurate modeling
involves the application of hetero-bifunctional cross-linkers incorporating
fast-acting photoactivatable diazirines coupled with conventional
amine-targeting N-hydroxysuccinimide esters, and
we demonstrate that conventional homo-bifunctional reagents used in
cross-linking kinetically trap dynamic states in the ensemble. Therefore,
the application of both classes of cross-linker provides an opportunity
to empirically detect protein dynamics during integrative structural
modeling.