Hedgehog proteins create a dynamic cholesterol interface.

During formation of the Hedgehog (Hh) signaling proteins, cooperative activities of the Hedgehog INTein (Hint) fold and Sterol Recognition Region (SRR) couple autoproteolysis to cholesterol ligation. The cholesteroylated Hh morphogens play essential roles in embryogenesis, tissue regeneration, and tumorigenesis. Despite the centrality of cholesterol in Hh function, the full structure of the Hint-SRR ("Hog") domain that attaches cholesterol to the last residue of the active Hh morphogen remains enigmatic. In this work, we combine molecular dynamics simulations, photoaffinity crosslinking, and mutagenesis assays to model cholesterolysis intermediates in the human Sonic Hedgehog (hSHH) protein. Our results provide evidence for a hydrophobic Hint-SRR interface that forms a dynamic, non-covalent cholesterol-Hog complex. Using these models, we suggest a unified mechanism by which Hh proteins can recruit, sequester, and orient cholesterol, and offer a molecular basis for the effects of disease-causing hSHH mutations.

Publisher Correction: Dual roles of the sterol recognition region in Hedgehog protein modification.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Dual roles of the sterol recognition region in Hedgehog protein modification.

Nature provides a number of mechanisms to encode dynamic information in biomolecules. In metazoans, there exist rare chemical modifications that occur in entirely unique regimes. One such example occurs in the Hedgehog (Hh) morphogens, proteins singular across all domains of life for the nature of their covalent ligation to cholesterol. The isoform- and context-specific efficiency of this ligation profoundly impacts the activity of Hh morphogens and represents an unexplored facet of Hh ligand-dependent cancers. To elucidate the chemical mechanism of this modification, we have defined roles of the uncharacterized sterol recognition region (SRR) in Hh proteins. We use a combination of sequence conservation, directed mutagenesis, and biochemical assays to specify residues of the SRR participate in cellular and biochemical aspects of Hh cholesterolysis. Our investigations offer a functional portrait of this region, providing opportunities to identify parallel reactivity in nature and a template to design tools in chemical biology.

Native top-down mass spectrometry provides insights into the copper centers of membrane-bound methane monooxygenase.

Aerobic methane oxidation is catalyzed by particulate methane monooxygenase (pMMO), a copper-dependent, membrane metalloenzyme composed of subunits PmoA, PmoB, and PmoC. Characterization of the copper active site has been limited by challenges in spectroscopic analysis stemming from the presence of multiple copper binding sites, effects of detergent solubilization on activity and crystal structures, and the lack of a heterologous expression system. Here we utilize nanodiscs coupled with native top-down mass spectrometry (nTDMS) to determine the copper stoichiometry in each pMMO subunit and to detect post-translational modifications (PTMs). These results indicate the presence of a mononuclear copper center in both PmoB and PmoC. pMMO-nanodisc complexes with a higher stoichiometry of copper-bound PmoC exhibit increased activity, suggesting that the PmoC copper site plays a role in methane oxidation activity. These results provide key insights into the pMMO copper centers and demonstrate the ability of nTDMS to characterize complex membrane-bound metalloenzymes.

Fluorescent gold nanoclusters for efficient cancer cell targeting.

Well-known surface properties of gold nanoparticles (AuNPs) offer easy surface modification with desired biomolecule, thus enabling them to be used for targeting and imaging of cancer cells/tissues. However, targeting and imaging capability come through after synthesis coating of AuNPs' surface with targeting or imaging molecules. Attempts have been made to conjugate both imaging and targeting molecules over the AuNPs, but have seen limited success. Hence, exploiting the fluorescence properties of gold nanoclusters (AuNCs), we have synthesized glucose-coated AuNCs for exhibiting both the imaging and targeting properties. These clusters have shown rapid and selective uptake in cancerous (A549) cells when compared with bovine serum albumin-coated AuNCs.

Cu+-specific CopB transporter: Revising P1B-type ATPase classification.

The copper-transporting P1B-ATPases, which play a key role in cellular copper homeostasis, have been divided traditionally into two subfamilies, the P1B-1-ATPases or CopAs and the P1B-3-ATPases or CopBs. CopAs selectively export Cu+ whereas previous studies and bioinformatic analyses have suggested that CopBs are specific for Cu2+ export. Biochemical and spectroscopic characterization of Sphaerobacter thermophilus CopB (StCopB) show that, while it does bind Cu2+, the binding site is not the prototypical P1B-ATPase transmembrane site and does not involve sulfur coordination as proposed previously. Most important, StCopB exhibits metal-stimulated ATPase activity in response to Cu+, but not Cu2+, indicating that it is actually a Cu+ transporter. X-ray absorption spectroscopic studies indicate that Cu+ is coordinated by four sulfur ligands, likely derived from conserved cysteine and methionine residues. The histidine-rich N-terminal region of StCopB is required for maximal activity, but is inhibitory in the presence of divalent metal ions. Finally, reconsideration of the P1B-ATPase classification scheme suggests that the P1B-1- and P1B-3-ATPase subfamilies both comprise Cu+ transporters. These results are completely consistent with the known presence of only Cu+ within the reducing environment of the cytoplasm, which should eliminate the need for a Cu2+ P1B-ATPase.

ATP-enhanced peroxidase-like activity of gold nanoparticles.

Gold nanoparticles (AuNPs) are known to possess intrinsic biological peroxidase-like activity that has applications in development of numerous biosensors. The reactivity of the Au atoms at the surface of AuNPs is critical to the performance of such biosensors, yet little is known about the effect of biomolecules and ions on the peroxidase-like activity. In this work, the effect of ATP and other biologically relevant molecules and ions over peroxidase-like activity of AuNPs are described. Contrary to the expectation that nanoparticles exposed to biomolecules may lose the catalytic property, ATP and ADP addition enhanced the peroxidase-like activity of AuNPs. The catalytic activity was unaltered by the addition of free phosphate, sulphate and carbonate anions however, addition of ascorbic acid to the reaction mixture diminished the intrinsic peroxidase-like activity of AuNPs, even in the presence of ATP and ADP. In contrast to AuNPs, ATP did not synergize and improve the peroxidase activity of the natural peroxidase enzyme, horseradish peroxidase.

YC-1 binding to the ß subunit of soluble guanylyl cyclase overcomes allosteric inhibition by the α subunit.

Soluble guanylate cyclase (sGC) is a heterodimeric heme protein and the primary nitric oxide receptor. NO binding stimulates cyclase activity, leading to regulation of cardiovascular physiology and making sGC an attractive target for drug discovery. YC-1 and related compounds stimulate sGC both independently and synergistically with NO and CO binding; however, where the compounds bind and how they work remain unknown. Using linked equilibrium binding measurements, surface plasmon resonance, and domain truncations in Manduca sexta and bovine sGC, we demonstrate that YC-1 binds near or directly to the heme-containing domain of the ß subunit. In the absence of CO, YC-1 binds with a Kd of 9-21 µM, depending on the construct. In the presence of CO, these values decrease to 0.6-1.1 µM. Pfizer compound 25 bound ∼10-fold weaker than YC-1 in the absence of CO, whereas compound BAY 41-2272 bound particularly tightly in the presence of CO (Kd = 30-90 nM). Additionally, we found that CO binds much more weakly to heterodimeric sGC proteins (Kd = 50-100 µM) than to the isolated heme domain (Kd = 0.2 µM for Manduca ß H-NOX/PAS). YC-1 greatly enhanced binding of CO to heterodimeric sGC, as expected (Kd ∼ 1 µM). These data indicate the α subunit induces a heme pocket conformation with a lower affinity for CO and NO. YC-1 family compounds bind near the heme domain, overcoming the α subunit effect and inducing a heme pocket conformation with high affinity. We propose this high-affinity conformation is required for the full-length protein to achieve high catalytic activity.

Crystal structure of the Alpha subunit PAS domain from soluble guanylyl cyclase.

Soluble guanylate cyclase (sGC) is a heterodimeric heme protein of ≈ 150 kDa and the primary nitric oxide receptor. Binding of NO stimulates cyclase activity, leading to regulation of cardiovascular physiology and providing attractive opportunities for drug discovery. How sGC is stimulated and where candidate drugs bind remains unknown. The α and ß sGC chains are each composed of Heme-Nitric Oxide Oxygen (H-NOX), Per-ARNT-Sim (PAS), coiled-coil and cyclase domains. Here, we present the crystal structure of the α1 PAS domain to 1.8 Å resolution. The structure reveals the binding surfaces of importance to heterodimer function, particularly with respect to regulating NO binding to heme in the ß1 H-NOX domain. It also reveals a small internal cavity that may serve to bind ligands or participate in signal transduction.