High-risk human papillomaviruses (HPVs) cause various cancers. While type-specific prophylactic vaccines are available, additional anti-viral strategies are highly desirable. Initial HPV cell entry involves receptor-switching induced by structural capsid modifications. These modifications are initiated by interactions with cellular heparan sulphates (HS), however, their molecular nature and functional consequences remain elusive. Combining virological assays with hydrogen/deuterium exchange mass spectrometry, and atomic force microscopy, we investigate the effect of capsid-HS binding and structural activation. We show how HS-induced structural activation requires a minimal HS-chain length and simultaneous engagement of several binding sites by a single HS molecule. This engagement introduces a pincer-like force that stabilizes the capsid in a conformation with extended capsomer linkers. It results in capsid enlargement and softening, thereby likely facilitating L1 proteolytic cleavage and subsequent L2-externalization, as needed for cell entry. Our data supports the further devising of prophylactic strategies against HPV infections.

• MeSH
• Heparitin Sulfate * metabolism   chemistry   MeSH
• Papillomavirus Infections virology   MeSH
• Virus Internalization * MeSH
• Capsid * metabolism   chemistry   MeSH
• Humans MeSH
• Human Papillomavirus Viruses MeSH
• Human papillomavirus 16 metabolism   physiology   MeSH
• Microscopy, Atomic Force * MeSH
• Papillomaviridae physiology   MeSH
• Polysaccharides metabolism   chemistry   MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Capsid Proteins * metabolism   chemistry   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Heparitin Sulfate * MeSH
• Polysaccharides MeSH
• Capsid Proteins * MeSH

Hydrogen/deuterium exchange (HDX) followed by mass spectrometry detection (MS) provides a fast, reliable, and detailed solution for the assessment of a protein structure. It has been widely recognized as an indispensable tool and already approved by several regulatory agencies as a structural technique for the validation of protein biopharmaceuticals, including antibody-based drugs. Antibodies are of a key importance in life and medical sciences but considered to be challenging analytical targets because of their compact structure stabilized by disulfide bonds and due to the presence of glycosylation. Despite these difficulties, there are already numerous excellent studies describing MS-based antibody structure characterization. In this chapter, we describe a universal HDX-MS workflow. Deeper attention is paid to sample handling, optimization procedures, and feasibility stages, as these elements of the HDX experiment are crucial for obtaining reliable detailed and spatially well-resolved information.

• Keywords
• Antibody, Biosimilars, Hydrogen/deuterium exchange, Mass spectrometry, Protein structure and dynamics, Proteolysis,
• MeSH
• Deuterium MeSH
• Mass Spectrometry MeSH
• Antibodies * MeSH
• Hydrogen Deuterium Exchange-Mass Spectrometry * MeSH
• Hydrogen MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Deuterium MeSH
• Antibodies * MeSH
• Hydrogen MeSH

Protein hydrogen/deuterium exchange (HDX) coupled to mass spectrometry (MS) can be used to study interactions of proteins with various ligands, to describe the effects of mutations, or to reveal structural responses of proteins to different experimental conditions. It is often described as a method with virtually no limitations in terms of protein size or sample composition. While this is generally true, there are, however, ligands or buffer components that can significantly complicate the analysis. One such compound, that can make HDX-MS troublesome, is DNA. In this chapter, we will focus on the analysis of protein-DNA interactions, describe the detailed protocol, and point out ways to overcome the complications arising from the presence of DNA.

• Keywords
• DNA, Hydrogen/deuterium exchange, Protein–DNA binding, Structural mass spectrometry, Transcription factor,
• MeSH
• Data Analysis MeSH
• Chromatography, Liquid MeSH
• DNA-Binding Proteins chemistry   metabolism   MeSH
• DNA chemistry   metabolism   MeSH
• Protein Interaction Domains and Motifs MeSH
• Humans MeSH
• Transcription Factors MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Hydrogen Deuterium Exchange-Mass Spectrometry * methods   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA-Binding Proteins MeSH
• DNA MeSH
• Transcription Factors MeSH

The natural function of cellobiose dehydrogenase (CDH) to donate electrons from its catalytic flavodehydrogenase (DH) domain via its cytochrome (CYT) domain to lytic polysaccharide monooxygenase (LPMO) is an example of a highly efficient extracellular electron transfer chain. To investigate the function of the CYT domain movement in the two occurring electron transfer steps, two CDHs from the ascomycete Neurospora crassa (NcCDHIIA and NcCDHIIB) and five chimeric CDH enzymes created by domain swapping were studied in combination with the fungus' own LPMOs (NcLPMO9C and NcLPMO9F). Kinetic and electrochemical methods and hydrogen/deuterium exchange mass spectrometry were used to study the domain movement, interaction, and electron transfer kinetics. Molecular docking provided insights into the protein-protein interface, the orientation of domains, and binding energies. We find that the first, interdomain electron transfer step from the catalytic site in the DH domain to the CYT domain depends on steric and electrostatic interface complementarity and the length of the protein linker between both domains but not on the redox potential difference between the FAD and heme b cofactors. After CYT reduction, a conformational change of CDH from its closed state to an open state allows the second, interprotein electron transfer (IPET) step from CYT to LPMO to occur by direct interaction of the b-type heme and the type-2 copper center. Chimeric CDH enzymes favor the open state and achieve higher IPET rates by exposing the heme b cofactor to LPMO. The IPET, which is influenced by interface complementarity and the heme b redox potential, is very efficient with bimolecular rates between 2.9 × 105 and 1.1 × 106 M-1 s-1.

• Publication type
• Journal Article MeSH

The limited information available on the structure of complexes involving transcription factors and cognate DNA response elements represents a major obstacle in the quest to understand their mechanism of action at the molecular level. We implemented a concerted structural proteomics approach, which combined hydrogen-deuterium exchange (HDX), quantitative protein-protein and protein-nucleic acid cross-linking (XL), and homology analysis, to model the structure of the complex between the full-length DNA binding domain (DBD) of Forkhead box protein O4 (FOXO4) and its DNA binding element (DBE). The results confirmed that FOXO4-DBD assumes the characteristic forkhead topology shared by these types of transcription factors, but its binding mode differs significantly from those of other members of the family. The results showed that the binding interaction stabilized regions that were rather flexible and disordered in the unbound form. Surprisingly, the conformational effects were not limited only to the interface between bound components, but extended also to distal regions that may be essential to recruiting additional factors to the transcription machinery. In addition to providing valuable new insights into the binding mechanism, this project provided an excellent evaluation of the merits of structural proteomics approaches in the investigation of systems that are not directly amenable to traditional high-resolution techniques.

• Keywords
• DNA, FOXO4, cross-linking, molecular modeling, protein, protein-nucleic acid cross-linking, trans-dichlorodiamineplatinum(II), hydrogen-deuterium exchange, transcription factor, transplatin,
• MeSH
• DNA-Binding Proteins chemistry   metabolism   MeSH
• DNA chemistry   metabolism   MeSH
• Mass Spectrometry MeSH
• Molecular Structure MeSH
• Response Elements MeSH
• Transcription Factors chemistry   metabolism   MeSH
• Deuterium Exchange Measurement MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• DNA-Binding Proteins MeSH
• DNA MeSH
• Transcription Factors MeSH

The heme-based oxygen sensor histidine kinase AfGcHK is part of a two-component signal transduction system in bacteria. O2 binding to the Fe(II) heme complex of its N-terminal globin domain strongly stimulates autophosphorylation at His183 in its C-terminal kinase domain. The 6-coordinate heme Fe(III)-OH- and -CN- complexes of AfGcHK are also active, but the 5-coordinate heme Fe(II) complex and the heme-free apo-form are inactive. Here, we determined the crystal structures of the isolated dimeric globin domains of the active Fe(III)-CN- and inactive 5-coordinate Fe(II) forms, revealing striking structural differences on the heme-proximal side of the globin domain. Using hydrogen/deuterium exchange coupled with mass spectrometry to characterize the conformations of the active and inactive forms of full-length AfGcHK in solution, we investigated the intramolecular signal transduction mechanisms. Major differences between the active and inactive forms were observed on the heme-proximal side (helix H5), at the dimerization interface (helices H6 and H7 and loop L7) of the globin domain and in the ATP-binding site (helices H9 and H11) of the kinase domain. Moreover, separation of the sensor and kinase domains, which deactivates catalysis, increased the solvent exposure of the globin domain-dimerization interface (helix H6) as well as the flexibility and solvent exposure of helix H11. Together, these results suggest that structural changes at the heme-proximal side, the globin domain-dimerization interface, and the ATP-binding site are important in the signal transduction mechanism of AfGcHK. We conclude that AfGcHK functions as an ensemble of molecules sampling at least two conformational states.

• Keywords
• bacterial protein kinase, crystal structure, globin, heme-containing oxygen sensor, histidine kinase, hydrogen-deuterium exchange, signal transduction, two component signal transduction system,
• MeSH
• Bacterial Proteins chemistry   metabolism   MeSH
• Phosphorylation MeSH
• Heme chemistry   MeSH
• Histidine Kinase chemistry   metabolism   MeSH
• Mass Spectrometry MeSH
• Crystallography, X-Ray MeSH
• Protein Structure, Quaternary MeSH
• Oxygen metabolism   MeSH
• Models, Molecular MeSH
• Myxococcales metabolism   MeSH
• Oxidation-Reduction MeSH
• Protein Domains MeSH
• Signal Transduction MeSH
• Deuterium Exchange Measurement MeSH
• Ferric Compounds chemistry   MeSH
• Ferrous Compounds chemistry   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Heme MeSH
• Histidine Kinase MeSH
• Oxygen MeSH
• Ferric Compounds MeSH
• Ferrous Compounds MeSH