Tara Sprules

A Ubl/ubiquitin switch in the activation of Parkin

Mutations in Parkin and PINK1 cause an inherited early‐onset form of Parkinsons disease. The two proteins function together in a mitochondrial quality control pathway whereby PINK1 accumulates on damaged mitochondria and activates Parkin to induce mitophagy. How PINK1 kinase activity releases the auto‐inhibited ubiquitin ligase activity of Parkin remains unclear. Here, we identify a binding switch between phospho‐ubiquitin (pUb) and the ubiquitin‐like domain (Ubl) of Parkin as a key element. By mutagenesis and SAXS, we show that pUb binds to RING1 of Parkin at a site formed by His302 and Arg305. pUb binding promotes disengagement of the Ubl from RING1 and subsequent Parkin phosphorylation. A crystal structure of Parkin Δ86–130 at 2.54 Å resolution allowed the design of mutations that specifically release the Ubl domain from RING1. These mutations mimic pUb binding and promote Parkin phosphorylation. Measurements of the E2 ubiquitin‐conjugating enzyme UbcH7 binding to Parkin and Parkin E3 ligase activity suggest that Parkin phosphorylation regulates E3 ligase activity downstream of pUb binding.

Competing allosteric mechanisms modulate substrate binding in a dimeric enzyme

Allostery has been studied for many decades, yet it remains challenging to determine experimentally how it occurs at a molecular level. We have developed an approach combining isothermal titration calorimetry, circular dichroism and nuclear magnetic resonance spectroscopy to quantify allostery in terms of protein thermodynamics, structure and dynamics. This strategy was applied to study the interaction between aminoglycoside N-(6′)-acetyltransferase-Ii and one of its substrates, acetyl coenzyme A. It was found that homotropic allostery between the two active sites of the homodimeric enzyme is modulated by opposing mechanisms. One follows a classical Koshland-Némethy-Filmer (KNF) paradigm, whereas the other follows a recently proposed mechanism in which partial unfolding of the subunits is coupled to ligand binding. Competition between folding, binding and conformational changes represents a new way to govern energetic communication between binding sites.

The substrate-bound crystal structure of a Baeyer-Villiger monooxygenase exhibits a Criegee-like conformation.

The Baeyer–Villiger monooxygenases (BVMOs) are a family of bacterial flavoproteins that catalyze the synthetically useful Baeyer–Villiger oxidation reaction. This involves the conversion of ketones into esters or cyclic ketones into lactones by introducing an oxygen atom adjacent to the carbonyl group. The BVMOs offer exquisite regio- and enantiospecificity while acting on a wide range of substrates. They use only NADPH and oxygen as cosubstrates, and produce only NADP+ and water as byproducts, making them environmentally attractive for industrial purposes. Here, we report the first crystal structure of a BVMO, cyclohexanone monooxygenase (CHMO) from Rhodococcus sp. HI-31 in complex with its substrate, cyclohexanone, as well as NADP+ and FAD, to 2.4 Å resolution. This structure shows a drastic rotation of the NADP+ cofactor in comparison to previously reported NADP+-bound structures, as the nicotinamide moiety is no longer positioned above the flavin ring. Instead, the substrate, cyclohexanone, is found at this location, in an appropriate position for the formation of the Criegee intermediate. The rotation of NADP+ permits the substrate to gain access to the reactive flavin peroxyanion intermediate while preventing it from diffusing out of the active site. The structure thus reveals the conformation of the enzyme during the key catalytic step. CHMO is proposed to undergo a series of conformational changes to gradually move the substrate from the solvent, via binding in a solvent excluded pocket that dictates the enzyme’s chemospecificity, to a location above the flavin–peroxide adduct where catalysis occurs.

Apoptotic Regulation by MCL-1 through Heterodimerization

Myeloid cell leukemia 1 (MCL-1), an anti-apoptotic BCL-2 family member active in the preservation of mitochondrial integrity during apoptosis, has fundamental roles in development and hematopoiesis and is dysregulated in human cancers. It bears a unique, intrinsically unstructured, N-terminal sequence, which leads to its instability in cells and hinders protein production and structural characterization. Here, we present collective data from NMR spectroscopy and titration calorimetry to reveal the selectivity of MCL-1 in binding BCL-2 homology 3 (BH3) ligands of interest for mammalian biology. The N-terminal sequence weakens the BH3 interactions but does not affect selectivity. Its removal by calpain-mediated limited proteolysis results in a stable BCL-2-like core domain of MCL-1 (cMCL-1). This core is necessary and sufficient for BH3 ligand binding. Significantly, we also characterized the in vitro protein-protein interaction between cMCL-1 and activated BID by size exclusion chromatography and NMR titrations. This interaction occurs in a very slow manner in solution but is otherwise similar to the interaction between cMCL-1 and BID-BH3 peptides. We also present the solution structure of complex cMCL-1·hBID-BH3, which completes the family portrait of MCL-1 complexes and may facilitate drug discovery against human tumors.

NMR solution structure of ImB2, a protein conferring immunity to antimicrobial activity of the type IIa bacteriocin, carnobacteriocin B2.

Bacteriocins produced by lactic acid bacteria are potent antimicrobial compounds which are active against closely related bacteria. Producer strains are protected against the effects of their cognate bacteriocins by immunity proteins that are located on the same genetic locus and are coexpressed with the gene encoding the bacteriocin. Several structures are available for class IIa bacteriocins; however, to date, no structures are available for the corresponding immunity proteins. We report here the NMR solution structure of the 111-amino acid immunity protein for carnobacteriocin B2 (ImB2). ImB2 folds into a globular domain in aqueous solution which contains an antiparallel four-helix bundle. Extensive packing by hydrophobic side chains in adjacent helices forms the core of the protein. The C-terminus, containing a fifth helix and an extended strand, is held against the four-helix bundle by hydrophobic interactions with helices 3 and 4. Most of the charged and polar residues in the protein face the solvent. Helix 3 is well-defined to residue 55, and a stretch of nascent helix followed by an unstructured loop joins it to helix 4. No interaction is observed between ImB2 and either carnobacteriocin B2 (CbnB2) or its precursor. Protection from the action of CbnB2 is only observed when ImB2 is expressed within the cell. The loop between helices 3 and 4, and a hydrophobic pocket which it partially masks, may be important for interaction with membrane receptors responsible for sensitivity to class IIa bacteriocins.

Proteolytically Stable Cancer Targeting Peptides with High Affinity for Breast Cancer Cells

Cancer cell targeting peptides have emerged as a highly efficient approach for selective delivery of chemotherapeutics and diagnostics to different cancer cells. However, the use of α-peptides in pharmaceutical applications is hindered by their enzymatic degradation and low bioavailability. Starting with a 10-mer α-peptide 18 that we developed previously, here we report three novel analogues of 18 that are proteolytically stable and display better (up to 3.5-fold) affinity profiles for breast cancer cells compared to 18. The design strategy involved replacement of two or three amino acids in the sequence of 18 with d-residues or β(3)-amino acids. Such replacement maintained the specificity for cancer cells (MDA-MB-435, MDA-MB-231, and MCF-7) with low affinity for control noncancerous cells (MCF-10A and HUVEC), showed an increase in secondary structure, and rendered the analogues completely stable to human serum and liver homogenate from mice. The three analogues are potentially safe with minimal cellular toxicity and are efficient targeting moieties for specific drug delivery to breast cancer cells. The strategy used here may be adapted to develop peptide analogues that will target other cancer cell types.

Analyzing protein folding cooperativity by differential scanning calorimetry and NMR spectroscopy.

Some marginally stable proteins undergo microsecond time scale folding reactions that involve significant populations of partly ordered forms, making it difficult to discern individual steps in their folding pathways. It has been suggested that many of these proteins fold non-cooperatively, with no significant barriers to separate the energy landscape into distinct thermodynamic states. Here we present an approach for studying the cooperativity of rapid protein folding with a combination of differential scanning calorimetry (DSC), nuclear magnetic resonance (NMR) relaxation dispersion experiments, and an analysis of the temperature dependence of amide (1)H and (15)N chemical shifts. We applied this method to the PBX homeodomain (PBX-HD), which folds on the microsecond time scale and produces a broad DSC thermogram with an elevated and steeply sloping native-state heat capacity baseline, making it a candidate for barrierless folding. However, by globally fitting the NMR thermal melt and DSC data, and by comparing these results to those obtained from the NMR relaxation dispersion experiments, we show that the native form of the protein undergoes two-state exchange with a small population of the thermally denatured form, well below the melting temperature. This result directly demonstrates the coexistence of distinct folded and unfolded forms and firmly establishes that folding of PBX-HD is cooperative. Further, we see evidence of large-scale structural and dynamical changes within the native state by NMR, which helps to explain the broad and shallow DSC profile. This study illustrates the potential of combining calorimetry with NMR dynamics experiments to dissect mechanisms of protein folding.

Nuclear magnetic resonance solution structure of PisI, a group B immunity protein that provides protection against the type IIa bacteriocin piscicolin 126, PisA.

Lactic acid bacteria produce and secrete bacteriocins. These bacteriocins are potent antimicrobial peptides that are active against other closely related bacteria. As a means of self-protection, producer organisms also express immunity proteins. Immunity proteins are generally located on the same genetic locus and are cotranscribed with the bacteriocin. Although some cross immunity between bacteriocins has been observed, immunity proteins are typically highly specific. Immunity proteins for the type IIa bacteriocins range from 81 to 115 amino acids in length and display substantial variation in their sequences. Nonetheless, such immunity proteins have been classified into three groupings (groups A, B, and C) according to sequence homology. The structures of a group C (ImB2) and two group A (EntA-im and PedB) immunity proteins have previously been reported. We herein report the nuclear magnetic resonance solution structure of the remaining class of the type IIa immunity proteins. PisI, a 98-amino acid protein, is a group B immunity protein conferring immunity against piscicolin 126 (PisA). Like ImB2, EntA-im, and PedB, PisI folds into a globular protein in aqueous solution and contains an antiparallel four-helix bundle. Compared to ImB2 and EntA-im, PisI has a substantially longer and more flexible N-terminus, but a shorter C-terminus. No direct interaction between the bacteriocin and immunity protein is observed by NMR in either aqueous or membrane mimicking environments. This further suggests that the mechanism that mediates immunity is not due to a direct bacteriocin-immunity protein interaction but rather is receptor-mediated. It has now been confirmed that the four-helix bundle is indeed a structural motif among the type IIa immunity proteins.

Small-Molecule Ligands of GD2 Ganglioside, Designed from NMR Studies, Exhibit Induced-Fit Binding and Bioactivity

Ganglioside GD2 is a cell surface glycosphingolipid. Targeting of GD2, i.e., by anti-GD2 mAb 3F8, is used clinically for cancer diagnosis, prognosis, and therapy. Here, the conformations of free GD2, and of GD2 bound to mAb 3F8, were resolved by saturation transfer difference NMR and molecular modeling. Then, three small-molecule cyclic peptide ligands that bind to GD2 selectively were designed. Transferred nuclear Overhauser enhancement of the GD2-bound conformation of the peptide ligands showed an induced-fit binding mechanism. The mAb 3F8 and the peptidic GD2 ligands mediate similar biological functions in cell-based assays of calcium fluxes and src activation. Thus, small molecules can selectively and functionally interact with a sugar head group. This work furthers the concept of rationally designing ligands for carbohydrate targets, and may be expanded to other clinically relevant gangliosides.

1H, 13C and 15N resonance assignments of the bb′ domains of human protein disulfide isomerase

Protein disulfide isomerase (PDI) participates in protein folding and catalyses formation of disulfide bonds. The b′ domain of human PDI contributes to binding unfolded proteins; its structure is stabilized by the b domain. Here, we report NMR chemical shift assignments for the bb′ fragment.