David VanderVelde

Structures of the Sgt2/SGTA Dimerization Domain with the Get5/UBL4A UBL Domain Reveal an Interaction that Forms a Conserved Dynamic Interface

In the cytoplasm, the correct delivery of membrane proteins is an essential and highly regulated process. The posttranslational targeting of the important tail-anchor membrane (TA) proteins has recently been under intense investigation. A specialized pathway, called the guided entry of TA proteins (GET) pathway in yeast and the transmembrane domain recognition complex (TRC) pathway in vertebrates, recognizes endoplasmic-reticulum-targeted TA proteins and delivers them through a complex series of handoffs. An early step is the formation of a complex between Sgt2/SGTA, a cochaperone with a presumed ubiquitin-like-binding domain (UBD), and Get5/UBL4A, a ubiquitin-like domain (UBL)-containing protein. We structurally characterize this UBD/UBL interaction for both yeast and human proteins. This characterization is supported by biophysical studies that demonstrate that complex formation is mediated by electrostatics, generating an interface that has high-affinity with rapid kinetics. In total, this work provides a refined model of the interplay of Sgt2 homologs in TA targeting.

Proton–hydride tautomerism in hydrogen evolution catalysis

Significance The discovery of efficient hydrogen evolution catalysts for solar fuels production continues to be an active research field. Catalyst optimization depends on detailed knowledge of the elementary chemical reaction steps involved in catalysis. Isolation of intermediates in catalytic processes is uncommon owing to their necessarily low stability. By using weak acids, we have isolated and characterized an intermediate in the 2e− + 2H+ → H2 reaction catalyzed by η5-pentamethylcyclopentadienyl (Cp*) Rh(κ2-2,2′-bipyridyl) [Rh(bpy)]. We find that the preferred site of Cp*Rh(bpy) protonation is not the metal center but is the Cp* ligand. Despite the reputation of Cp* as a stable ligand in organometallic chemistry, these results suggest an important role for close metal–ligand cooperation in promoting hydrogen–evolution catalysis. Efficient generation of hydrogen from renewable resources requires development of catalysts that avoid deep wells and high barriers. Information about the energy landscape for H2 production can be obtained by chemical characterization of catalytic intermediates, but few have been observed to date. We have isolated and characterized a key intermediate in 2e– + 2H+ → H2 catalysis. This intermediate, obtained by treatment of Cp*Rh(bpy) (Cp*, η5-pentamethylcyclopentadienyl; bpy, κ2-2,2′-bipyridyl) with acid, is not a hydride species but rather, bears [η4-Cp*H] as a ligand. Delivery of a second proton to this species leads to evolution of H2 and reformation of η5-Cp* bound to rhodium(III). With suitable choices of acids and bases, the Cp*Rh(bpy) complex catalyzes facile and reversible interconversion of H+ and H2.

Nitrogen Insertion into a Corrole Ring: Iridium Monoazaporphyrins†

A new route to rare porphyrinoids: The non-innocence of the corrole ring allows the oxidative ring insertion of a nitrogen atom under mild conditions (see scheme; NBS=N-bromosuccinimide). The resulting meso-substituted azaporphyrins exhibit high-energy Soret absorption bands and red luminescence. This new synthetic route will allow for the development of novel azaporphyrin complexes with relevance to the study of biomimetic oxidations.

Get5 Carboxyl-terminal Domain is a Novel Dimerization Motif that Tethers an Extended Get4/Get5 Complex

Background: The Get4/Get5 protein complex is a homodimer mediated by the Get5 carboxyl domain. Results: The Get5 homodimerization motif forms a structurally conserved helical domain allowing Get4/Get5 to adopt an extended solution conformation. Conclusion: Get5 homodimerization is mediated by a 35-residue sequence stabilized by a few conserved hydrophobic interactions. Significance: The Get5 carboxyl domain contains a novel example of a stable dimerization motif. Tail-anchored trans-membrane proteins are targeted to membranes post-translationally. The proteins Get4 and Get5 form an obligate complex that catalyzes the transfer of tail-anchored proteins destined to the endoplasmic reticulum from Sgt2 to the cytosolic targeting factor Get3. Get5 forms a homodimer mediated by its carboxyl domain. We show here that a conserved motif exists within the carboxyl domain. A high resolution crystal structure and solution NMR structures of this motif reveal a novel and stable helical dimerization domain. We additionally determined a solution NMR structure of a divergent fungal homolog, and comparison of these structures allows annotation of specific stabilizing interactions. Using solution x-ray scattering and the structures of all folded domains, we present a model of the full-length Get4/Get5 complex.

Biofilm-specific extracellular matrix proteins of nontypeable Haemophilus influenzae.

Nontypeable Haemophilus influenzae (NTHi), a human respiratory tract pathogen, can form colony biofilms in vitro. Bacterial cells and the amorphous extracellular matrix (ECM) constituting the biofilm can be separated using sonication. The ECM from 24- and 96-h NTHi biofilms contained polysaccharides and proteinaceous components as detected by nuclear magnetic resonance (NMR) and Fourier transform infrared spectroscopy (FTIR) spectroscopy. More conventional chemical assays on the biofilm ECM confirmed the presence of these components and also DNA. Proteomics revealed eighteen proteins present in biofilm ECM that were not detected in planktonic bacteria. One ECM protein was unique to 24-h biofilms, two were found only in 96-h biofilms, and fifteen were present in the ECM of both 24- and 96-h NTHi biofilms. All proteins identified were either associated with bacterial membranes or cytoplasmic proteins. Immunocytochemistry showed two of the identified proteins, a DNA-directed RNA polymerase and the outer membrane protein OMP P2, associated with bacteria and biofilm ECM. Identification of biofilm-specific proteins present in immature biofilms is an important step in understanding the in vitro process of NTHi biofilm formation. The presence of a cytoplasmic protein and a membrane protein in the biofilm ECM of immature NTHi biofilms suggests that bacterial cell lysis may be a feature of early biofilm formation.

An initial event in the insect innate immune response: structural and biological studies of interactions between β-1,3-glucan and the N-terminal domain of β-1,3-glucan recognition protein.

In response to invading microorganisms, insect β-1,3-glucan recognition protein (βGRP), a soluble receptor in the hemolymph, binds to the surfaces of bacteria and fungi and activates serine protease cascades that promote destruction of pathogens by means of melanization or expression of antimicrobial peptides. Here we report on the nuclear magnetic resonance (NMR) solution structure of the N-terminal domain of βGRP (N-βGRP) from Indian meal moth (Plodia interpunctella), which is sufficient to activate the prophenoloxidase (proPO) pathway resulting in melanin formation. NMR and isothermal calorimetric titrations of N-βGRP with laminarihexaose, a glucose hexamer containing β-1,3 links, suggest a weak binding of the ligand. However, addition of laminarin, a glucose polysaccharide (~6 kDa) containing β-1,3 and β-1,6 links that activates the proPO pathway, to N-βGRP results in the loss of NMR cross-peaks from the backbone (15)N-(1)H groups of the protein, suggesting the formation of a large complex. Analytical ultracentrifugation (AUC) studies of formation of the N-βGRP-laminarin complex show that ligand binding induces self-association of the protein-carbohydrate complex into a macro structure, likely containing six protein and three laminarin molecules (~102 kDa). The macro complex is quite stable, as it does not undergo dissociation upon dilution to submicromolar concentrations. The structural model thus derived from this study for the N-βGRP-laminarin complex in solution differs from the one in which a single N-βGRP molecule has been proposed to bind to a triple-helical form of laminarin on the basis of an X-ray crystallographic structure of the N-βGRP-laminarihexaose complex [Kanagawa, M., Satoh, T., Ikeda, A., Adachi, Y., Ohno, N., and Yamaguchi, Y. (2011) J. Biol. Chem. 286, 29158-29165]. AUC studies and phenoloxidase activation measurements conducted with the designed mutants of N-βGRP indicate that electrostatic interactions involving Asp45, Arg54, and Asp68 between the ligand-bound protein molecules contribute in part to the stability of the N-βGRP-laminarin macro complex and that a decreased stability is accompanied by a reduced level of activation of the proPO pathway. An increased level of β-1,6 branching in laminarin also results in destabilization of the macro complex. These novel findings suggest that ligand-induced self-association of the βGRP-β-1,3-glucan complex may form a platform on a microbial surface for recruitment of downstream proteases, as a means of amplification of the initial signal of pathogen recognition for the activation of the proPO pathway.

The dormancy specific regulator, SutA, is an intrinsically-disordered protein that modulates transcription initiation in Pseudomonas aeruginosa

SutA is upregulated during growth arrest in Pseudomonas aeruginosa and binds RNA polymerase (RNAP), causing widespread changes in gene expression. Using biochemical, structural and genetic methods, we examined how SutA interacts with RNAP and the functional consequences of these interactions. SutA consists of a central α-helix with unstructured N and C-terminal tails. It binds to the β1 domain of RNAP and competes with DNA, leading to effects that are either activating or repressing, depending on the sigma (σ) factor and promoter. Our data suggest that SutA is unlike conventional DNA-binding transcription factors, in that interactions between its α-helix and RNAP allow its acidic N-terminal tail to modulate the path of DNA within the transcription initiation complex, while its C-terminal tail stabilizes its interaction with RNAP. These activities help enhance expression of diverse genes, including essential ones such as the ribosomal RNA operons, under conditions of long-term resource limitation.

Lewis Acid Enhancement of Proton Induced CO2 Cleavage: Bond Weakening and Ligand Residence Time Effects

Though Lewis acids (LAs) have been shown to have profound effects on carbon dioxide (CO2) reduction catalysis, the underlying cause of the improved reactivity remains unclear. Herein, we report a well-defined molecular system for probing the role of LA additives in the reduction of CO2 to carbon monoxide (CO) and water. Mo(0) CO2 complex (2) forms adducts with a series of LAs, demonstrating CO2 activation that correlates linearly with the strength of the LA. Protons induce C-O cleavage of these LA adducts, in contrast to the CO2 displacement primarily observed in the absence of LA. CO2 cleavage shows dependence on both bond activation and the residence time of the bound small molecule, demonstrating the influence of both kinetic and thermodynamic factors on promoting productive CO2 reduction chemistry.

Modulating the Folding Landscape of Superoxide Dismutase 1 with Targeted Molecular Binders

Amyotrophic lateral sclerosis, or Lou Gehrigs disease, is characterized by motor neuron death, with average survival times of two to five years. One cause of this disease is the misfolding of superoxide dismutaseu20051 (SOD1), a phenomenon influenced by point mutations spanning the protein. Herein, we used an epitope-specific high-throughput screen to identify a peptide ligand that stabilizes the SOD1 native conformation and accelerates its folding by a factor of 2.5. This strategy may be useful for fundamental studies of protein energy landscapes as well as designing new classes of therapeutics.