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Summary: Native mass spectrometry (MS) is a powerful technique for studying noncovalent protein-protein interactions. Here, native MS was employed to examine the noncovalent interactions involved in homodimerization of antibody half molecules (HL) in hinge-deleted human IgG4 (IgG4Δhinge). By analyzing the concentration dependence of the relative distribution of monomer HL and dimer (HL)2 species, the apparent dissociation constant (KD) for this interaction was determined. In combination with site-directed mutagenesis, the relative contributions of residues at the CH3-CH3 interface to this interaction could be characterized and corresponding KD values quantified over a range of 10−10–10−4 M. The critical importance of this noncovalent interaction in maintaining the intact dimeric structure was also proven for the full-length IgG4 backbone. Using time-resolved MS, the kinetics of the interaction could be measured, reflecting the dynamics of IgG4 HL exchange. Hence, native MS has provided a quantitative view of local structural features that define biological properties of IgG4. [ABSTRACT FROM AUTHOR]

Summary: P pili are important adhesive fibers involved in kidney infection by uropathogenic Escherichia coli. Pilus subunits are characterized by a large groove resulting from lack of a β strand. Polymerization of pilus subunits occurs via the donor-strand exchange (DSE) mechanism initiated when the N terminus of an incoming subunit interacts with the P5 region/pocket of the previously assembled subunit groove. Here, we solve the structure of the PapD:PapF complex in order to understand why PapF undergoes slow DSE. The structure reveals that the PapF P5 pocket is partially obstructed. MD simulations show this region of PapF is flexible compared with its equivalent in PapH, a subunit that also has an obstructed P5 pocket and is unable to undergo DSE. Using electrospray-ionization mass spectrometry, we show that mutations in the P5 region result in increased DSE rates. Thus, partial obstruction of the P5 pocket serves as a modulating mechanism of DSE. [Copyright &y& Elsevier]