Characterization of the family of Mistic homologues

BACKGROUND: Mistic is a unique Bacillus subtilis protein with virtually no detectable homologues in GenBank, which appears to integrate into the bacterial membrane despite an overall hydrophilic composition. These unusual properties have been shown to be useful for high-yield recombinant expression of other membrane proteins through fusion to the C-terminus of Mistic. To better understand the structure and function of Mistic, we systematically searched for and characterized homologous proteins among closely related bacteria. RESULTS: Three homologues of Mistic were found with 62% to 93% residue identity, all only 84 residues in length, corresponding to the C-terminal residues of B. subtilis Mistic. In every case, the Mistic gene was found partially overlapping a downstream gene for a K(+ )channel protein. Residue variation amongst these sequences is restricted to loop regions of the protein's structure, suggesting that secondary structure elements and overall fold have been conserved. Additionally, all three homologues retain the functional ability to chaperone fusion partners to the membrane. CONCLUSION: The functional core of Mistic consists of 84 moderately conserved residues that are sufficient for membrane targeting and integration. Understanding the minimal structural and chemical complexity of Mistic will lead to insights into the mechanistic underpinnings of Mistic-chaperoned membrane integration, as well as how to optimize its use for the recombinant heterologous expression of other integral membrane proteins of interest

Neogenin Interacts with Hemojuvelin through Its Two Membrane-Proximal Fibronectin Type III Domains

Hemojuvelin is a recently identified iron-regulatory protein that plays an important role in affecting the expression of hepcidin, a key iron regulatory hormone. Although the underlying mechanism of this process is not clear, several hemojuvelin-binding proteins, including the cell surface receptor neogenin and bone morphogenetic protein (BMP) cytokines, have been identified. The ectodomain of neogenin is composed of four immunoglobulin-like (Ig) domains followed by six fibronectin type III-like (FNIII) domains. Here we report expression of soluble versions of hemojuvelin and neogenin for biochemical characterization of their interaction and the interaction of HJV with BMP-2. Hemojuvelin normally undergoes an autocatalytic cleavage, and as in vivo, recombinant hemojuvelin exists as a mixture of cleaved and uncleaved forms. Neogenin binds to cleaved and noncleaved hemojuvelin, as verified by its binding to an uncleaved mutant hemojuvelin. We localized the hemojuvelin binding site on neogenin to the membrane-proximal fifth and sixth FNIII domains and the juxtamembrane linker and showed that a fragment containing only this region binds 2–3 orders of magnitude more tightly than the entire neogenin ectodomain. Binding to the most membrane-proximal region of neogenin may play a role in regulating the levels of soluble and membrane-bound forms of hemojuvelin, which in turn would influence the amount of free BMP-2 available for binding to its receptors and triggering transcription of the hepcidin gene. Our finding that BMP-2 and neogenin bind simultaneously to hemojuvelin raises the possibility that neogenin is part of a multiprotein complex at the hepatocyte membrane involving BMP, its receptors, and hemojuvelin

Designer TGFβ Superfamily Ligands with Diversified Functionality

Transforming Growth Factor – beta (TGFβ) superfamily ligands, including Activins, Growth and Differentiation Factors (GDFs), and Bone Morphogenetic Proteins (BMPs), are excellent targets for protein-based therapeutics because of their pervasiveness in numerous developmental and cellular processes. We developed a strategy termed RASCH (Random Assembly of Segmental Chimera and Heteromer), to engineer chemically-refoldable TGFβ superfamily ligands with unique signaling properties. One of these engineered ligands, AB208, created from Activin-βA and BMP-2 sequences, exhibits the refolding characteristics of BMP-2 while possessing Activin-like signaling attributes. Further, we find several additional ligands, AB204, AB211, and AB215, which initiate the intracellular Smad1-mediated signaling pathways more strongly than BMP-2 but show no sensitivity to the natural BMP antagonist Noggin unlike natural BMP-2. In another design, incorporation of a short N-terminal segment from BMP-2 was sufficient to enable chemical refolding of BMP-9, without which was never produced nor refolded. Our studies show that the RASCH strategy enables us to expand the functional repertoire of TGFβ superfamily ligands through development of novel chimeric TGFβ ligands with diverse biological and clinical values

Preparation and Characterization of Stable α-Synuclein Lipoprotein Particles

Multiple neurodegenerative diseases are caused by the aggregation of the human α-Synuclein (α-Syn(6)) protein. α-Syn possesses high structural plasticity and the capability of interacting with membranes. Both features are not only essential for its physiological function but also play a role in the aggregation process. Recently it has been proposed that α-Syn is able to form lipid-protein particles reminiscent of high-density lipoproteins. Here, we present a method to obtain a stable and homogeneous population of nanometer-sized particles composed of α-Syn and anionic phospholipids. These particles are called α-Syn lipoprotein (nano)particles to indicate their relationship to high-density lipoproteins formed by human apolipoproteins in vivo and of in vitro self-assembling phospholipid bilayer nanodiscs. Structural investigations of the α-Syn lipoprotein particles by circular dichroism (CD) and magic angle solid-state nuclear magnetic resonance (MAS SS-NMR) spectroscopy establish that α-Syn adopts a helical secondary structure within these particles. Based on cryo-electron microscopy (cryo-EM) and dynamic light scattering (DLS) α-Syn lipoprotein particles have a defined size with a diameter of ~23 nm. Chemical cross-linking in combination with solution-state NMR and multiangle static light scattering (MALS) of α-Syn particles reveal a high-order protein-lipid entity composed of approximately 8-10 α-Syn molecules. The close resemblance in size between cross-linked in vitro-derived α-Syn lipoprotein particles and a cross-linked species of endogenous α-Syn from SH-SY5Y human neuroblastoma cells indicates a potential functional relevance of α-Syn lipoprotein nanoparticles

2007 Annual progress report synopsis of the Center for Structures of Membrane Proteins

A synopsis of the 2007 annual progress report for the Center for Structures of Membrane Proteins, a specialized center of the Protein Structure Initiative

The Desensitization Gating of the MthK K+ Channel Is Governed by Its Cytoplasmic Amino Terminus

The RCK-containing MthK channel undergoes two inactivation processes: activation-coupled desensitization and acid-induced inactivation. The acid inactivation is mediated by the C-terminal RCK domain assembly. Here, we report that the desensitization gating is governed by a desensitization domain (DD) of the cytoplasmic N-terminal 17 residues. Deletion of DD completely removes the desensitization, and the process can be fully restored by a synthetic DD peptide added in trans. Mutagenesis analyses reveal a sequence-specific determinant for desensitization within the initial hydrophobic segment of DD. Proton nuclear magnetic resonance (1H NMR) spectroscopy analyses with synthetic peptides and isolated RCK show interactions between the two terminal domains. Additionally, we show that deletion of DD does not affect the acid-induced inactivation, indicating that the two inactivation processes are mutually independent. Our results demonstrate that the short N-terminal DD of MthK functions as a complete moveable module responsible for the desensitization. Its interaction with the C-terminal RCK domain may play a role in the gating process

BMP-2/6 Heterodimer Is More Effective than BMP-2 or BMP-6 Homodimers as Inductor of Differentiation of Human Embryonic Stem Cells

Bone Morphogenetic Protein (BMP) signaling pathways are involved in differentiation of stem cells into diverse cell types, and thus BMPs can be used as main guidance molecules for in vitro differentiation of human stem cells.We have analyzed the ability for inducing differentiation of the heterodimer BMP-2/BMP-6 (BMP-2/6) compared to the homodimers BMP-2 or BMP-6, using human embryonic stem (hES) cells H9 as model system. When incubated in a medium with high concentration of basic fibroblastic growth factor (FGF2), 100 ng/ml of human recombinant BMPs induced morphological changes and differentiation of hES cells in 24 to 48 hours. After 5 days, expression of differentiation markers was induced and quantified by quantitative PCR (qPCR) and flow cytometry. BMP-2/6 exhibited stronger activity for the induction of the expression of trophectodermal (CDX2) and endodermal (SOX17, GATA4, AFP) markers than BMP-2 or BMP-6 homodimers. BMP-2/6 also induced the expression of BMPR2 gene more effectively than BMP-2 or BMP-6 when used at the same concentration and time. Moreover, the percentage of cells expressing the surface endodermal marker CXCR4 was also increased for the heterodimer when compared to both homodimers. BMP-2/6 was a more potent activator of Smad-dependent (SMAD1/5) and Smad-independent signaling (mitogen-activated protein kinases ERK and p38) than BMP-2 and BMP-6, and the activation of these pathways might play a role in its increased potency for inducing hES cell differentiation.Therefore, we conclude that BMP-2/6 is more potent than BMP-2 or BMP-6 for inducing differentiation of hES cells, and it can be used as a more powerful substitute of these BMPs in in vitro differentiation guidance

In Situ Formation of an Azo Bridge on Proteins Controllable by Visible Light

Optical modulation of proteins provides superior spatiotemporal resolution for understanding biological processes, and photoswitches built on light-sensitive proteins have been significantly advancing neuronal and cellular studies. Small molecule photoswitches could complement protein-based switches by mitigating potential interference and affording high specificity for modulation sites. However, genetic encodability and responsiveness to nonultraviolet light, two desired properties possessed by protein photoswitches, are challenging to be engineered into small molecule photoswitches. Here we developed a small molecule photoswitch that can be genetically installed onto proteins in situ and controlled by visible light. A pentafluoro azobenzene-based photoswitchable click amino acid (F-PSCaa) was designed to isomerize in response to visible light. After genetic incorporation into proteins via the expansion of the genetic code, F-PSCaa reacts with a nearby cysteine within the protein generating an azo bridge in situ. The resultant bridge is switchable by visible light and allows conformation and binding of CaM to be regulated by such light. This photoswitch should prove valuable in optobiology for its minimal interference, site flexibility, genetic encodability, and response to the more biocompatible visible light