Tobias S. Ulmer

Many cytosolic proteins critical to membrane trafficking and function contain an unstructured domain that can bind to specific membranes, with a transition into an amphipathic helix induced upon membrane association. These inducible amphipathic helices often play a critical role in organelle recognition and subsequent function, but the tools used to characterize affinity towards specific membranes are low-throughput and dependent on the solubility of the inducible amphipathic helix. Here, we introduce a modular recombinant protein platform for rapidly measuring the binding affinity of inducible amphipathic helices towards a variety of membrane compositions and curvatures using high-throughput fluorescence anisotropy measurements. Inducible amphipathic helices are solubilized with a fluorescently-tagged small ubiquitin-like modifier (SUMO) protein and binding to membranes quantified by leveraging the unexpected decrease in fluorescence anisotropy upon binding, a phenomenon previously observed but not well understood. By using fluorescence anisotropy decay measurements, solution NMR experiments, and solution FRET, we deduce that this phenomenon likely occurs due to the local increase in fluorophore motion upon binding to the membrane enabled by vesicle membrane charge under low-salt conditions. This recombinant protein platform can be readily applied to any inducible amphipathic helix of interest, allowing for investigation of specific membrane biochemical parameters facilitating binding.

In biological membranes, proteins face a fundamentally different environment than in water. To avoid untenable lipid contacts with polar backbone atoms, they use the continuous hydrogen bonding achieved by α-helices or β-barrels to traverse membranes. Here, we show that integrin αX, and by homology αM, undermine this paradigm by partially unfolding the N-terminal third of their transmembrane (TM) helix. Unfolding results in a dynamic, frayed helix that weakens the association with its partnering β2 subunit to lower the activation threshold of integrin αXβ2-mediated cell adhesion. The extent of unfolding depends on membrane geometry, thereby establishing a mechanism for sensing membrane properties. The combination of adhesive control with sensory capacity in integrin αXβ2 and αMβ2 may achieve membrane localization-dependent receptor activation in leukocyte phagocytosis. The unfolding of the αX TM helix arises from a high number of α-helix-destabilizing residues that TM helices in general approach but do not exceed. Accordingly, backbone dynamics of TM helices may disrupt hydrogen bonds, modulate protein function, and optimize TM helix rigidity.

Many cytosolic proteins critical to membrane trafficking and function contain an unstructured domain that can bind to specific membranes, with a transition into an amphipathic helix induced upon membrane association. These inducible amphipathic helices often play a critical role in organelle recognition and subsequent function by these cytosolic proteins, but the tools and techniques used to characterize affinity towards specific membranes are low-throughput and highly dependent on the solubility of the inducible amphipathic helix. Here, we introduce a modular recombinant protein platform for rapidly measuring the binding affinity of inducible amphipathic helices towards a variety of membrane compositions and curvatures using high-throughput fluorescence anisotropy measurements. Inducible amphipathic helices are solubilized with a fluorescently tagged small ubiquitin-like modifier (SUMO) protein and binding to membranes quantified by leveraging the unexpected decrease in fluorescence anisotropy upon binding, a phenomenon previously observed but not well understood. By using fluorescence anisotropy decay measurements and solution NMR experiments, we deduce that this phenomenon likely occurs due to the local increase in fluorophore motion upon binding to the membrane. Altogether, this recombinant protein platform can be readily applied to any inducible amphipathic helix of interest, allowing for detailed investigation of the specific membrane biochemical parameters facilitating binding.

In the innate immune system, the CD33 receptor modulates microglial activity. Its downregulation promises to slow Alzheimer's disease, and it is already targeted in blood cancers. The mechanism underlying CD33 signaling is unresolved. Starting from the available crystal structure of its extracellular IgV-IgC1 domains, we have assembled a model of the human CD33 receptor by characterizing the oligomerization and structure of IgC1, transmembrane, and cytosolic domains in solution. IgC1 homodimerizes via intermolecular β-strand pairing and packing. In contrast, the 21-residue transmembrane helix of CD33 appears monomeric and straight, with a conserved thin neck and thick belly appearance followed by a positively charged cytosolic patch. The cytosolic domain is dynamically unstructured. Sequence alignment and AlphaFold models indicate that IgC domains in the family of human Siglecs, to which CD33 belongs, are surprisingly variable. Only Siglec-6 is identified to analogously dimerize via IgC1. Our CD33 structural model suggests that the receptor is not signaling via a monomer-dimer shift. Rather, we propose that, aided but also constrained by dimerization, multivalent ligands may concentrate the receptor transmembrane and cytosolic domains sufficiently to trigger colocalization with an activating kinase.

Vpr binding protein (VprBP), also known as DDB1- and CUL4-associated factor1 (DCAF1), is a recently identified atypical kinase and plays an important role in downregulating the transcription of tumor suppressor genes as well as increasing the risk for colon and prostate cancers. Melanoma is the most aggressive form of skin cancer arising from pigment-producing melanocytes and is often associated with the dysregulation of epigenetic factors targeting histones. Here, we demonstrate that VprBP is highly expressed and phosphorylates threonine 120 (T120) on histone H2A to drive the transcriptional inactivation of growth-regulatory genes in melanoma cells. As is the case for its epigenetic function in other types of cancers, VprBP acts to induce a gene silencing program dependent on H2AT120 phosphorylation (H2AT120p). The significance of VprBP-mediated H2AT120p is further underscored by the fact that VprBP knockdown- or VprBP inhibitor-induced lockage of H2AT120p mitigates melanoma tumor growth in xenograft models. Collectively, our results establish VprBP-mediated H2AT120p as a key epigenetic signal for melanomagenesis and suggest the therapeutic potential of targeting VprBP kinase activity for effective melanoma treatment.

Membrane proteins are commonly reconstituted in membrane mimics exhibiting discontinuous lipid bilayers. In contrast, the continuous membranes of cells are conceptually best represented by large unilamellar vesicles (LUVs). Here, we compared the thermodynamic stability of the integrin αIIbβ3 transmembrane (TM) complex between vesicles and bicelles to assess the consequence of this simplification. In LUVs, we further evaluated the strength of the αIIb(G972S)-β3(V700T) interaction that corresponds to the hydrogen bond interaction postulated for β2 integrins. An upper limit of 0.9 kcal/mol was estimated for superior TM complex stabilization in LUVs relative to bicelles. Compared to the αIIbβ3 TM complex stability in LUVs of 5.6 ± 0.2 kcal/mol, this limit is modest, indicating that bicelles performed well relative to LUVs. The implementation of β3(V700T) alleviated αIIb(G972S) destabilization by 0.4 ± 0.2 kcal/mol in confirmation of relatively weak hydrogen bonding. Interestingly, the hydrogen bond adjusts the TM complex stability to a level that is not achievable by merely varying the residue corresponding to αIIb(Gly972).

The ability to quantify protein–protein interactions without adding labels to protein has made isothermal titration calorimetry (ITC) a preferred technique to study proteins in aqueous solution. Here, we describe the application of ITC to the study of protein–protein interactions in membrane mimics using the association of integrin αIIb and β3 transmembrane domains in phospholipid bicelles as an example. A higher conceptual and experimental effort compared to water-soluble proteins is required for membrane proteins and rewarded with rare thermodynamic insight into this central class of proteins.

Structural diversity in α-helical membrane proteins (MP) arises from variations in helix-helix crossings and contacts that may bias amino acid usage. Here, we reveal systematic changes in transmembrane amino acid frequencies (f) as a function of the number of helices (n). For eukarya, breaks in f(n) trends of packing (Ala, Gly and Pro), polar, and hydrophobic residues identify different MP assembly principles for 2≤n≤7, 8≤n≤12 and n≥13. In bacteria, the first f break already occurs after n = 6 in correlation to an earlier n peak in MP size distribution and dominance of packing over polar interactions. In contrast to the later n brackets, the integration levels of helix bundles continuously increased in the first, most populous brackets indicating the formation of single structural units (domains). The larger first bracket of eukarya relates to a balance of polar and packing interactions that enlarges helix-helix combinatorial possibilities (MP diversity). Between the evolutionary old, packing and new, polar residues f anti-correlations extend over all biological taxa, broadly ordering them according to evolutionary history and allowing f estimates for the earliest forms of life. Next to evolutionary history, the amino acid composition of MP is determined by size (n), proteome diversity, and effective amino acid cost.

MMP-9 plays a direct role in the activation of pro-osteoclastogenic genes by cleaving histone H3N-terminal tail (H3NT) and altering chromatin architecture. Although H3 acetylation at K18 has been shown to stimulate MMP-9 enzymatic activity toward H3NT, nothing is known about the influence of other H3NT modifications on this epigenetic reaction. We show that H3 monomethylation at lysine 27 (H3K27me1) is essential for MMP-9-dependent H3NT proteolysis during RANKL-induced osteoclast differentiation. Through the recognition of H3K27me1 mark, MMP-9 localizes and generates H3NT proteolysis at the genes encoding osteoclast differentiation factors. By using RNAi and small molecule inhibitor approaches, we also confirmed that G9a is the major methyltransferase to catalyze H3K27me1 for MMP-9-dependent H3NT proteolysis and trigger the expression of osteoclast-specific genes. Our data establish new functions for G9a-mediated H3K27me1 in MMP-9-dependent H3NT proteolysis and demonstrate how histone modification can be exploited to regulate osteoclastogenic gene expression at the molecular level. Further studies are warranted to investigate the detailed mechanism by which G9a overexpression with concomitant dysregulation of osteoclastogenesis contributes to the pathogenesis of bone disorders.