Mycobacterium tuberculosis (Mtb)-specific γ9δ2 T cells secrete GzmA protective against intracellular Mtb growth. However, GzmA enzymatic activity is unnecessary for pathogen inhibition and the mechanisms of GzmA-mediated protection remain unknown. We show GzmA homodimerization is essential for opsonization of mycobacteria, altered uptake into human monocytes and subsequent pathogen clearance within the phagolysosome. While monomeric and homodimeric GzmA bind mycobacteria, only homodimers also bind CD14 and TLR4. Without access to surface expressed CD14 and TLR4, GzmA fails to inhibit intracellular Mtb. Upregulation of Rab11FIP1, was associated with inhibitory activity. Further, GzmA colocalized with and was regulated by protein disulfide isomerase (PDI)A1, which cleaves GzmA homodimers into monomers and prevents Mtb inhibitory activity. These studies identify previously unrecognized role for homodimeric GzmA structure in opsonization, phagocytosis and elimination of Mtb in human monocytes, and highlights PDIA1 as a potential host-directed therapy for prevention and treatment of tuberculosis, a major human disease.

Oxidative stress contributes to thrombosis in atherosclerosis, inflammation, infection, aging, and malignancy. Oxidant-induced cysteine modifications, including sulfenylation, can act as a redox-sensitive switch that controls protein function. Protein disulfide isomerase (PDI) is a prothrombotic enzyme with exquisitely redox-sensitive active-site cysteines.

Atypical hemolytic uremic syndrome (aHUS) is a rare thrombotic microangiopathy. Genetic variants in complement proteins are found in ≈60% of patients. Of these patients, ≈15% carry mutations in complement factor I (CFI). Factor I (FI) is a multidomain serine protease that cleaves and thereby inactivates C3b and C4b in the presence of cofactor proteins. Crystal structures have shown that FI possesses 2 calcium-binding domains, low-density lipoprotein receptor class A (LDLRA) 1 and LDLRA2. Yet, the role of calcium in FI is unknown. We determined that 9 genetic variants identified in aHUS (N151S, G162D, G188A, V230E, A240G, G243R, C247G, A258T, and Q260D) cluster around the calcium-binding site of LDLRA1. Using site-directed mutagenesis, we established that the synthesis of all, except A258T, was impaired, implying defective protein folding, perhaps due to loss of calcium binding. To further explore this possibility, we generated 12 alanine mutants that coordinate with the calcium in LDLRA1 and LDLRA2 (K239A, D242A, I244A, D246A, D252A, E253A, Y276A, N279A, E281A, D283A, D289A, and D290A) and are expected to perturb calcium binding. Except for K239A and Y276A, none of the mutants was secreted. These observations suggest that calcium ions play key structural and functional roles in FI.

Antiphospholipid antibodies targeting β-glycoprotein I (βGPI) cause thrombosis and pregnancy morbidity in antiphospholipid syndrome (APS) patients. How these antibodies recognize βGPI remains controversial.

Disulfide bonds drive protein correct folding, prevent protein aggregation, and stabilize three-dimensional structures of proteins and their assemblies. Dysregulation of this activity leads to several disorders, including cancer, neurodegeneration, and thrombosis. A family of 20+ enzymes, called thiol-isomerases (TIs), oversee this process in the endoplasmic reticulum of human cells to ensure efficacy and accuracy. While the biophysical and biochemical properties of cysteine residues are well-defined, our structural knowledge of how TIs select, interact and process their substrates remains poorly understood. How TIs structurally and functionally respond to changes in redox environment and other post-translational modifications remain unclear, too. We recently developed a workflow for site-specific incorporation of non-canonical amino acids into protein disulfide isomerase (PDI), the prototypical member of TIs. Combined with click chemistry, this strategy enabled us to perform single-molecule biophysical studies of PDI under various solution conditions. This paper details protocols and discusses challenges in performing these experiments. We expect this approach, combined with other emerging technologies in single-molecule biophysics and structural biology, to facilitate the exploration of the mechanisms by which TIs carry out their fascinating but poorly understood roles in humans, especially in the context of thrombosis.