Physical Chemistry of Cellular Liquid-Phase Separation.

Compartmentalization of biochemical processes is essential for cell function. Although membrane-bound organelles are well studied in this context, recent work has shown that phase separation is a key contributor to cellular compartmentalization through the formation of liquid-like membraneless organelles (MLOs). In this Minireview, the key mechanistic concepts that underlie MLO dynamics and function are first briefly discussed, including the relevant noncovalent interaction chemistry and polymer physical chemistry. Next, a few examples of MLOs and relevant proteins are given, along with their functions, which highlight the relevance of the above concepts. The developing area of active matter and non-equilibrium systems, which can give rise to unexpected effects in fluctuating cellular conditions, are also discussed. Finally, our thoughts for emerging and future directions in the field are discussed, including in vitro and in vivo studies of MLO physical chemistry and function.

EF5 Is the High-Affinity Mg(2+) Site in ALG-2.

The penta-EF-hand (PEF) protein ALG-2 (apoptosis-linked gene 2) has been implicated in several important physiological processes, including endoplasmic reticulum-Golgi vesicular transport and endosomal biogenesis/transport. ALG-2 was recently shown to harbor a metal ion-binding site with a high affinity for Mg(2+) and a low affinity for Ca(2+). We herein present the X-ray structure of Mg(2+)-bound ALG-2des23(wt). Although the C(α) trace is nearly indistinguishable from that of the Ca(2+)-free protein, the orientation of the C-terminal helix differs in the two structures. Consistent with that observation, replacement of the +x ligand in EF5, D169, with alanine eliminates high-affinity Mg(2+) binding. It also eliminates the low-affinity Ca(2+) site and lowers the affinity of the remaining Ca(2+)-binding sites, EF3 and EF1. The coordination environment in EF5 approaches ideal Mg(2+) octahedral geometry. The ligand array, consisting of three carboxylates (+x, +y, +z), a backbone carbonyl (-y), and two water molecules (-x, -z), may offer a recipe for a high-affinity, high-selectivity Mg(2+)-binding site. Sequence data for other PEF proteins indicate that select calpain large subunits, notably CAPN1 and CAPN8, may also possess a high-affinity Mg(2+)-binding site. In Mg(2+)-bound ALG-2, the carbonyl of F188 and the C-terminal carboxylate of V191 interact with the ε-ammonium group of K137 in the opposing subunit, suggesting that Mg(2+) binding could have an impact on dimerization. Interestingly, EF1 and EF3 are also occupied in the crystal, despite having modest affinity for Mg(2+). The results of a calorimetry-based analysis indicate that their Mg(2+) binding constants are 2 orders of magnitude lower than that determined for EF5.

Membrane Anchoring of α-Helical Proteins: Role of Tryptophan.

The function of membrane proteins relies on a defined orientation of protein relative to lipid. In apparent correlation to protein anchoring, tryptophan residues are enriched in the lipid headgroup region. To characterize the thermodynamic and structural basis of this relationship in α-helical membrane proteins, we examined the role of three conserved tryptophans in the folding of the heterodimeric integrin αIIbß3 transmembrane (TM) complex in phospholipid bicelles and mammalian membranes. In the homogenous lipid environment of bicelles, tryptophan was replaceable by residues of distinct polarities. The appropriate polarity was guided by the electrostatic potential of the tryptophan surrounding, suggesting that tryptophan can complement diverse environments by adjusting the orientation of its anisotropic side chain to achieve site-specific anchoring. As a sole membrane anchor, tryptophan made a contribution of 0.4 kcal/mol to TM complex stability in bicelles. In membranes, it proved more difficult to replace tryptophan even by tyrosine, indicating a superior capacity to interact with heterogeneous lipids of biological membranes. Interestingly, at intracellular TM helix ends, where integrin activation is initiated, sequence motifs that interact with lipids via opposing polarity patterns were found to restrict TM helix orientations beyond tryptophan anchoring. In contrast to bicelles, phenylalanine became the least accepted substitute in membranes, demonstrating an increased role of the hydrophobic effect. Altogether, our study implicates a wide amphiphilic range of tryptophan, membrane complexity, and the hydrophobic effect to be important factors in tryptophan membrane anchoring.