Structure of the Human VPS33B/VPS16B Complex
In humans, variants affecting expression/function of VPS33B or VPS16B (VIPAS39) can cause arthrogryposis, renal dysfunction and cholestasis (ARC) syndrome, a rare neonatal lethal condition with symptoms affecting many organ systems. These proteins form a VPS33B/VPS16B complex which is required for many cellular processes, including the production by bone marrow megakaryocytes of protein-carrying alpha granules, which are absent in the platelets of ARC syndrome patients. Previous students in the Kahr Lab studied the VPS33B/VPS16B complex by a variety of means, including negative-staining electron microscopy (EM), which yielded the dimensions and overall shape of a complex with two lobes resembling a dumbbell. However, key aspects of the complex including the molecular weight, composition and subunit arrangement remained elusive. My work demonstrates that human VPS33B/VPS16B is a pentameric complex containing two molecules of VPS33B and 3 of VPS16B. Negative staining EM imaging of avidin-labeled complex showed the VPS33B subunits present in opposing lobes, oriented in trans. Analysis of the effects of VPS16B truncations revealed that a central domain of this subunit is required for stable assembly of the complex. This work significantly advanced knowledge of the structure and composition of VPS33B/VPS16B, providing details that point to key insights into the potential cellular functions of this complex. I also developed a reliable, scalable and easy-to-modify expression system for recombinant human VPS33B/VPS16B, a library of mutated and truncated complexes, and a TEV-protease cleavable version of VPS33B that will support future research into VPS33B/VPS16B function.
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