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.

Structural insights into the interaction between adenovirus C5 hexon and human lactoferrin.

Adenovirus (AdV) infection of the respiratory epithelium is common but poorly understood. Human AdV species C types, such as HAdV-C5, utilize the Coxsackie-adenovirus receptor (CAR) for attachment and subsequently integrins for entry. CAR and integrins are however located deep within the tight junctions in the mucosa where they would not be easily accessible. Recently, a model for CAR-independent AdV entry was proposed. In this model, human lactoferrin (hLF), an innate immune protein, aids the viral uptake into epithelial cells by mediating interactions between the major capsid protein, hexon, and yet unknown host cellular receptor(s). However, a detailed understanding of the molecular interactions driving this mechanism is lacking. Here, we present a new cryo-EM structure of HAdV-5C hexon at high resolution alongside a hybrid structure of HAdV-5C hexon complexed with human lactoferrin (hLF). These structures reveal the molecular determinants of the interaction between hLF and HAdV-C5 hexon. hLF engages hexon primarily via its N-terminal lactoferricin (Lfcin) region, interacting with hexon's hypervariable region 1 (HVR-1). Mutational analyses pinpoint critical Lfcin contacts and also identify additional regions within hLF that critically contribute to hexon binding. Our study sheds more light on the intricate mechanism by which HAdV-C5 utilizes soluble hLF/Lfcin for cellular entry. These findings hold promise for advancing gene therapy applications and inform vaccine development. IMPORTANCE: Our study delves into the structural aspects of adenovirus (AdV) infections, specifically HAdV-C5 in the respiratory epithelium. It uncovers the molecular details of a novel pathway where human lactoferrin (hLF) interacts with the major capsid protein, hexon, facilitating viral entry, and bypassing traditional receptors such as CAR and integrins. The study's cryo-EM structures reveal how hLF engages hexon, primarily through its N-terminal lactoferricin (Lfcin) region and hexon's hypervariable region 1 (HVR-1). Mutational analyses identify critical Lfcin contacts and other regions within hLF vital for hexon binding. This structural insight sheds light on HAdV-C5's mechanism of utilizing soluble hLF/Lfcin for cellular entry, holding promise for gene therapy and vaccine development advancements in adenovirus research.

Cryo-EM structures of Trypanosoma brucei gambiense ISG65 with human complement C3 and C3b and their roles in alternative pathway restriction.

African Trypanosomes have developed elaborate mechanisms to escape the adaptive immune response, but little is known about complement evasion particularly at the early stage of infection. Here we show that ISG65 of the human-infective parasite Trypanosoma brucei gambiense is a receptor for human complement factor C3 and its activation fragments and that it takes over a role in selective inhibition of the alternative pathway C5 convertase and thus abrogation of the terminal pathway. No deposition of C4b, as part of the classical and lectin pathway convertases, was detected on trypanosomes. We present the cryo-electron microscopy (EM) structures of native C3 and C3b in complex with ISG65 which reveal a set of modes of complement interaction. Based on these findings, we propose a model for receptor-ligand interactions as they occur at the plasma membrane of blood-stage trypanosomes and may facilitate innate immune escape of the parasite.

A multifaceted strategy to improve recombinant expression and structural characterisation of a Trypanosoma invariant surface protein.

Identification of a protein minimal fragment amenable to crystallisation can be time- and labour intensive especially if large amounts are required and the protein has a complex fold and functionally important post-translational modifications. In addition, a lack of homologues and structural information can further complicate the design of a minimal expression construct. Recombinant expression in E. coli promises high yields, low costs and fast turnover times, but falls short for many extracellular, eukaryotic proteins. Eukaryotic expression systems provide an alternative but are costly, slow and require special handling and equipment. Using a member of a structurally uncharacterized, eukaryotic receptor family as an example we employ hydrogen-deuterium exchange mass spectrometry (HDX-MS) guided construct design in conjunction with truncation scanning and targeted expression host switching to identify a minimal expression construct that can be produced with high yields and moderate costs.