Mass spectrometry-complemented molecular modeling predicts the interaction interface for a camelid single-domain antibody targeting the Plasmodium falciparum circumsporozoite protein's C-terminal domain.

Background: Bioanalytical methods that enable rapid and high-detail characterization of binding specificities and strengths of protein complexes with low sample consumption are highly desired. The interaction between a camelid single domain antibody (sdAbCSP1) and its target antigen (PfCSP-Cext) was selected as a model system to provide proof-of-principle for the here described methodology. Research design and methods: The structure of the sdAbCSP1 - PfCSP-Cext complex was modeled using AlphaFold2. The recombinantly expressed proteins, sdAbCSP1, PfCSP-Cext, and the sdAbCSP1 - PfCSP-Cext complex, were subjected to limited proteolysis and mass spectrometric peptide analysis. ITEM MS (Intact Transition Epitope Mapping Mass Spectrometry) and ITC (Isothermal Titration Calorimetry) were applied to determine stoichiometry and binding strength. Results: The paratope of sdAbCSP1 mainly consists of its CDR3 (aa100-118). PfCSP-Cext's epitope is assembled from its α-helix (aa40-52) and opposing loop (aa83-90). PfCSP-Cext's GluC cleavage sites E46 and E58 were shielded by complex formation, confirming the predicted epitope. Likewise, sdAbCSP1's tryptic cleavage sites R105 and R108 were shielded by complex formation, confirming the predicted paratope. ITEM MS determined the 1:1 stoichiometry and the high complex binding strength, exemplified by the gas phase dissociation reaction enthalpy of 50.2 kJ/mol. The in-solution complex dissociation constant is 5 × 10-10 M. Conclusions: Combining AlphaFold2 modeling with mass spectrometry/limited proteolysis generated a trustworthy model for the sdAbCSP1 - PfCSP-Cext complex interaction interface.

Beyond the VSG layer: Exploring the role of intrinsic disorder in the invariant surface glycoproteins of African trypanosomes.

In the bloodstream of mammalian hosts, African trypanosomes face the challenge of protecting their invariant surface receptors from immune detection. This crucial role is fulfilled by a dense, glycosylated protein layer composed of variant surface glycoproteins (VSGs), which undergo antigenic variation and provide a physical barrier that shields the underlying invariant surface glycoproteins (ISGs). The protective shield's limited permeability comes at the cost of restricted access to the extracellular host environment, raising questions regarding the specific function of the ISG repertoire. In this study, we employ an integrative structural biology approach to show that intrinsically disordered membrane-proximal regions are a common feature of members of the ISG super-family, conferring the ability to switch between compact and elongated conformers. While the folded, membrane-distal ectodomain is buried within the VSG layer for compact conformers, their elongated counterparts would enable the extension beyond it. This dynamic behavior enables ISGs to maintain a low immunogenic footprint while still allowing them to engage with the host environment when necessary. Our findings add further evidence to a dynamic molecular organization of trypanosome surface antigens wherein intrinsic disorder underpins the characteristics of a highly flexible ISG proteome to circumvent the constraints imposed by the VSG coat.

A high-throughput target-based screening approach for the identification and assessment of Mycobacterium tuberculosis mycothione reductase inhibitors.

The World Health Organization's goal to combat tuberculosis (TB) is hindered by the emergence of anti-microbial resistance, therefore necessitating the exploration of new drug targets. Multidrug regimens are indispensable in TB therapy as they provide synergetic bactericidal effects, shorten treatment duration, and reduce the risk of resistance development. The research within our European RespiriTB consortium explores Mycobacterium tuberculosis energy metabolism to identify new drug candidates that synergize with bedaquiline, with the aim of discovering more efficient combination drug regimens. In this study, we describe the development and validation of a luminescence-coupled, target-based assay for the identification of novel compounds inhibiting Mycobacterium tuberculosis mycothione reductase (MtrMtb), an enzyme with a role in the protection against oxidative stress. Recombinant MtrMtb was employed for the development of a highly sensitive, robust high-throughput screening (HTS) assay by coupling enzyme activity to a bioluminescent readout. Its application in a semi-automated setting resulted in the screening of a diverse library of ~130,000 compounds, from which 19 hits were retained after an assessment of their potency, selectivity, and specificity. The selected hits formed two clusters and four fragment molecules, which were further evaluated in whole-cell and intracellular infection assays. The established HTS discovery pipeline offers an opportunity to deliver novel MtrMtb inhibitors and lays the foundation for future efforts in developing robust biochemical assays for the identification and triaging of inhibitors from high-throughput library screens. IMPORTANCE: The growing anti-microbial resistance poses a global public health threat, impeding progress toward eradicating tuberculosis. Despite decades of active research, there is still a dire need for the discovery of drugs with novel modes of action and exploration of combination drug regimens. Within the European RespiriTB consortium, we explore Mycobacterium tuberculosis energy metabolism to identify new drug candidates that synergize with bedaquiline, with the aim of discovering more efficient combination drug regimens. In this study, we present the development of a high-throughput screening pipeline that led to the identification of M. tuberculosis mycothione reductase inhibitors.

Biophysical characterization of the Plasmodium falciparum circumsporozoite protein's N-terminal domain.

The circumsporozoite protein (CSP) is the main surface antigen of the Plasmodium sporozoite (SPZ) and forms the basis of the currently only licensed anti-malarial vaccine (RTS,S/AS01). CSP uniformly coats the SPZ and plays a pivotal role in its immunobiology, in both the insect and the vertebrate hosts. Although CSP's N-terminal domain (CSPN ) has been reported to play an important role in multiple CSP functions, a thorough biophysical and structural characterization of CSPN is currently lacking. Here, we present an alternative method for the recombinant production and purification of CSPN from Plasmodium falciparum (PfCSPN ), which provides pure, high-quality protein preparations with high yields. Through an interdisciplinary approach combining in-solution experimental methods and in silico analyses, we provide strong evidence that PfCSPN is an intrinsically disordered region displaying some degree of compaction.