Towards Probing Structure and Function Relationships of Proteins in Complex Sample Types Using HX-MS

The functional protein state is a dynamic one, and this behaviour should be accurately reported by our biophysical toolset. Hydrogen-exchange mass spectrometry (HX-MS) is a powerful means of probing changes in the conformational ensemble of interacting proteins, and helps shape our understanding of structure and function relationships. This dissertation describes the development of novel tools for HX-MS, geared towards interacting with biologically relevant systems that are prohibitively large and complex for current structural approaches. First, we introduced a set of standards to correct for dispensing during sample workup. This improved both systematic and random error, and increased the statistical power in differential experiments. Next, we scaled HX-compatible digestion strategies to determine how the analysis of complexes is limited. Digestion with traditional proteases was efficient, with modest coverage of a > 500 kDa sample. Our results suggested that the remaining limiting factors in the analysis of larger systems were related to chromatographic performance. We demonstrated the potential of specific prolyl-endoproteases to mitigate sample complexity. However, we discovered that peptide mapping was inadequate in all proteolytic approaches, and should be resolved if complete HX-MS datasets are desired. A proteomics-inspired nanoHX-MS system was next described. Resolution was improved by eliminating post-column band-broadening with our in-source configuration and a 50-fold improvement in sensitivity was achieved. We then investigated the validity of using overlapping peptides to increase structural resolution. Induction of secondary structure, and charge effects upon interaction with the chromatographic stationary phase perturbed exchange behaviour. Therefore, the fundamental assumption that residue exchange rates are independent of their parent peptide is invalid. These effects must be accounted for to obtain accurate modelling of site-resolved exchange with any high-resolution strategy. Finally, a multivariate strategy was tailored for large scale HX-MS screens. HX-MS readings were complemented by functional data (IC50) and used to characterize a panel of 18 compounds against Eg5, a mitotic kinesin. Canonical mechanisms were confirmed and roughly classified based on inhibitory strength. A modified binding mode and novel allosteric mechanism was discovered for Terpendole E, an inhibitor with activity in clinically relevant resistant Eg5 mutants

Structures and Dynamics of Native-State Transmembrane Protein Targets and Bound Lipids

Membrane proteins work within asymmetric bilayers of lipid molecules that are critical for their biological structures, dynamics and interactions. These properties are lost when detergents dislodge lipids, ligands and subunits, but are maintained in native nanodiscs formed using styrene maleic acid (SMA) and diisobutylene maleic acid (DIBMA) copolymers. These amphipathic polymers allow extraction of multicomponent complexes of post-translationally modified membrane-bound proteins directly from organ homogenates or membranes from diverse types of cells and organelles. Here, we review the structures and mechanisms of transmembrane targets and their interactions with lipids including phosphoinositides (PIs), as resolved using nanodisc systems and methods including cryo-electron microscopy (cryo-EM) and X-ray diffraction (XRD). We focus on therapeutic targets including several G protein-coupled receptors (GPCRs), as well as ion channels and transporters that are driving the development of next-generation native nanodiscs. The design of new synthetic polymers and complementary biophysical tools bodes well for the future of drug discovery and structural biology of native membrane:protein assemblies (memteins)

Lactoferrin binding protein B – a bi-functional bacterial receptor protein

<div><p>Lactoferrin binding protein B (LbpB) is a bi-lobed outer membrane-bound lipoprotein that comprises part of the lactoferrin (Lf) receptor complex in <i>Neisseria meningitidis</i> and other Gram-negative pathogens. Recent studies have demonstrated that LbpB plays a role in protecting the bacteria from cationic antimicrobial peptides due to large regions rich in anionic residues in the C-terminal lobe. Relative to its homolog, transferrin-binding protein B (TbpB), there currently is little evidence for its role in iron acquisition and relatively little structural and biophysical information on its interaction with Lf. In this study, a combination of crosslinking and deuterium exchange coupled to mass spectrometry, information-driven computational docking, bio-layer interferometry, and site-directed mutagenesis was used to probe LbpB:hLf complexes. The formation of a 1:1 complex of iron-loaded Lf and LbpB involves an interaction between the Lf C-lobe and LbpB N-lobe, comparable to TbpB, consistent with a potential role in iron acquisition. The Lf N-lobe is also capable of binding to negatively charged regions of the LbpB C-lobe and possibly other sites such that a variety of higher order complexes are formed. Our results are consistent with LbpB serving dual roles focused primarily on iron acquisition when exposed to limited levels of iron-loaded Lf on the mucosal surface and effectively binding apo Lf when exposed to high levels at sites of inflammation.</p></div

LbpB mutants.

<p>LbpB mutants.</p

Proposed functions of LbpB.

<p>(LEFT) LbpB may be involved in the iron-acquisition pathway. At low concentrations of holo-hLf, LbpB may use its LbpB-N binding mode to preferentially bind iron-loaded lactoferrin and shuttle it to LbpA, forming a ternary complex and hijacking the iron. (RIGHT) Cleavage of LbpB from the membrane may be dependent on the presence of high levels of hLf in the extracellular milieu or simply a constitutive property of <i>N</i>. <i>meningitidis</i> cells in the NalP phase-variable ON-state. The release of LbpB from the membrane is done in an effort to sequester lactoferricin, antibodies, and possibly form large lattices of hLf as to prevent proteolytical processing into its derivative cationic antimicrobial peptides.</p

Receptor lobe binding contributions in TbpB and LbpB.

<p>Cartoon representations of each recombinant LbpB protein are displayed beside their respective BLI steady-state binding curve from binding hLf. (A) Intact LbpB, K_{D app} = 72.8 ± 3.24nM. (B) LbpB-N lobe, K_{D app} = 126 ± 48nM. (C) LbpB-C lobe K_{D app} = 279 ± 15nM. C-lobe Hill slope was calculated to be 1.98 ± 0.13 implying positive cooperativity. (D) Intact-lgsm, K_{D app} = 140 ±82.4nM (E). LbpB-C lobe-lgsm had no observed binding.</p

Specificity of LbpB and TbpB for iron-loaded glycoprotein.

<p>(A) Competitive solid-phase binding assay of TbpB with apo/holo hTf and LbpB with apo/holo hLf. Recombinant MBP-TbpB (top two rows) and MBP-LbpB (bottom two rows) were applied to nitrocellulose paper, the paper blocked and then incubated with apo- or holo- glycoprotein overnight in a ¼ serially diluted fashion (A, 20nM; B, 5nM; C, 1.25nM; D, 0.31nM; E, 0.07nM; F, 0.01nM; G, 4.88 × 10^-3nM; H, 0nM). Iron-loaded HRP-conjugated glycoprotein (HRP-hTf or HRP-hLf) was then introduced into the binding mixture. Presence of a dot represents the displacement of any protein bound to TbpB or LbpB by the HRP-conjugate at the given concentration. (B) SDS-PAGE/affinity capture representing receptor protein (MBP-TbpB, 122kDa; or MBP-LbpB, 122kDa) captured by Sepharose resins conjugated to their cognate apo- or holo-glycoprotein (hTf-r, hLf-r).</p