The myelin sheath is a tightly packed multilayered membrane structure insulating selected axons in the central and the peripheral nervous systems. Myelin is a biochemically unique membrane, containing a specific set of proteins. In this study, we expressed and purified recombinant human myelin P2 protein and determined its crystal structure to a resolution of 1.85 Å. A fatty acid molecule, modeled as palmitate based on the electron density, was bound inside the barrel-shaped protein. Solution studies using synchrotron radiation indicate that the crystal structure is similar to the structure of the protein in solution. Docking experiments using the high-resolution crystal structure identified cholesterol, one of the most abundant lipids in myelin, as a possible ligand for P2, a hypothesis that was proven by fluorescence spectroscopy. In addition, electrostatic potential surface calculations supported a structural role for P2 inside the myelin membrane. The potential membrane-binding properties of P2 and a peptide derived from its N terminus were studied. Our results provide an enhanced view into the structure and function of the P2 protein from human myelin, which is able to bind both monomeric lipids inside its cavity and membrane surfaces.

Phytochromes are photoreceptors in plants, fungi, and various microorganisms and cycle between metastable red light-absorbing (Pr) and far-red light-absorbing (Pfr) states. Their light responses are thought to follow a conserved structural mechanism that is triggered by isomerization of the chromophore. Downstream structural changes involve refolding of the so-called tongue extension of the phytochrome-specific GAF-related (PHY) domain of the photoreceptor. The tongue is connected to the chromophore by conserved DIP and PRSF motifs and a conserved tyrosine, but the role of these residues in signal transduction is not clear. Here, we examine the tongue interactions and their interplay with the chromophore by substituting the conserved tyrosine (Tyr) in the phytochrome from the extremophile bacterium with phenylalanine. Using optical and FTIR spectroscopy, X-ray solution scattering, and crystallography of chromophore-binding domain (CBD) and CBD-PHY fragments, we show that the absence of the Tyr hydroxyl destabilizes the β-sheet conformation of the tongue. This allowed the phytochrome to adopt an α-helical tongue conformation regardless of the chromophore state, hence distorting the activity state of the protein. Our crystal structures further revealed that water interactions are missing in the Y263F mutant, correlating with a decrease of the photoconversion yield and underpinning the functional role of Tyr in phytochrome conformational changes. We propose a model in which isomerization of the chromophore, refolding of the tongue, and globular conformational changes are represented as weakly coupled equilibria. The results also suggest that the phytochromes have several redundant signaling routes.

Phytochrome proteins control the growth, reproduction, and photosynthesis of plants, fungi, and bacteria. Light is detected by a bilin cofactor, but it remains elusive how this leads to activation of the protein through structural changes. We present serial femtosecond X-ray crystallographic data of the chromophore-binding domains of a bacterial phytochrome at delay times of 1 ps and 10 ps after photoexcitation. The data reveal a twist of the D-ring, which leads to partial detachment of the chromophore from the protein. Unexpectedly, the conserved so-called pyrrole water is photodissociated from the chromophore, concomitant with movement of the A-ring and a key signaling aspartate. The changes are wired together by ultrafast backbone and water movements around the chromophore, channeling them into signal transduction towards the output domains. We suggest that the observed collective changes are important for the phytochrome photoresponse, explaining the earliest steps of how plants, fungi and bacteria sense red light.

Saltatory conduction of nerve impulses along axonal membranes depends on the presence of a multilayered membrane, myelin, that wraps around the axon. Myelin basic protein (MBP) and myelin protein 2 (P2) are intimately involved in the generation of the myelin sheath. They are also implicated in a number of neurological diseases, including autoimmune diseases of both the central and peripheral nervous systems. Here, we have used atomic force microsopy (AFM) to study the effects of MBP and P2 on lipid bilayers. MBP in association with a mica substrate appeared unstructured, and tended to coat the mica surface in the form of a monolayer. In contrast, P2 appeared as discrete particles, with molecular volumes consistent with the formation of both monomers and dimers. Either MBP or P2, at micromolar concentrations, caused stacking of brain lipid bilayers. This stacking effect was significantly potentiated when both proteins were added together. Bilayers composed of phosphatidylcholine (PC) and phosphatidylserine (PS) were stacked by MBP, provided that cholesterol was also present; in contrast, P2 did not stack PC/PS/cholesterol bilayers. Hence, the bilayer stacking effects of the two proteins have different lipid requirements.

The important role that extracellular adenosine plays in many physiological processes is mediated by the adenosine class of G protein-coupled receptors, a class of receptors that also responds to the antagonist caffeine, the most widely used pharmacological agent in the world. The crystallographic model of the human adenosine A(2A) receptor was recently solved to 2.6Å in complex with the antagonist ZM241385, which is also referred to as "super-caffeine" because of its strong antagonistic effect on adenosine receptors. The crystallographic model revealed some unexpected and unusual features of the adenosine A(2A) receptor structure that have led to new studies on the receptor and the re-examination of pre-existing data. Compared to other known GPCR structures, the adenosine A(2A) receptor has a unique ligand binding pocket that is nearly perpendicular to the membrane plane. The ligand binding site highlights the integral role of the helical core together with the extracellular loops and the four disulfide bridges in the extracellular domain, in ligand recognition by the adenosine class of GPCRs.