RBR E3 ubiquitin ligases: new structures, new insights, new questions

The RBR (RING-BetweenRING-RING) or TRIAD [two RING fingers and a DRIL (double RING finger linked)] E3 ubiquitin ligases comprise a group of 12 complex multidomain enzymes. This unique family of E3 ligases includes parkin, whose dysfunction is linked to the pathogenesis of early-onset Parkinson's disease, and HOIP (HOIL-1-interacting protein) and HOIL-1 (haem-oxidized IRP2 ubiquitin ligase 1), members of the LUBAC (linear ubiquitin chain assembly complex). The RBR E3 ligases share common features with both the larger RING and HECT (homologous with E6-associated protein C-terminus) E3 ligase families, directly catalysing ubiquitin transfer from an intrinsic catalytic cysteine housed in the C-terminal domain, as well as recruiting thioester-bound E2 enzymes via a RING domain. Recent three-dimensional structures and biochemical findings of the RBRs have revealed novel protein domain folds not previously envisioned and some surprising modes of regulation that have raised many questions. This has required renaming two of the domains in the RBR E3 ligases to more accurately reflect their structures and functions: the C-terminal Rcat (required-for-catalysis) domain, essential for catalytic activity, and a central BRcat (benign-catalytic) domain that adopts the same fold as the Rcat, but lacks a catalytic cysteine residue and ubiquitination activity. The present review discusses how three-dimensional structures of RBR (RING1-BRcat-Rcat) E3 ligases have provided new insights into our understanding of the biochemical mechanisms of these important enzymes in ubiquitin biology. INTRODUCTIO

Cytochrome c as a Peroxidase: Activation of the Precatalytic Native State by H

In addition to serving as respiratory electron shuttle, ferri-cytochrome c (cyt c) acts as a peroxidase; i.e., it catalyzes the oxidation of organic substrates by H2O2. This peroxidase function plays a key role during apoptosis. Typical peroxidases have a five-coordinate heme with a vacant distal coordination site that permits the iron center to interact with H2O2. In contrast, native cyt c is six-coordinate, as the distal coordination site is occupied by Met80. It thus seems counterintuitive that native cyt c would exhibit peroxidase activity. The current work scrutinizes the origin of this structure-function mismatch. Cyt c-catalyzed peroxidase reactions show an initial lag phase that is consistent with the in situ conversion of a precatalyst to an active peroxidase. Using mass spectrometry, we demonstrate the occurrence of cyt c self-oxidation in the presence of H2O2. The newly generated oxidized proteoforms are shown to possess significantly enhanced peroxidase activity. H2O2-induced modifications commence with oxidation of Tyr67, followed by permanent displacement of Met80 from the heme iron. The actual peroxidase activation step corresponds to subsequent side chain carbonylation, likely at Lys72/73. The Tyr67-oxidized/carbonylated protein has a vacant distal ligation site, and it represents the true peroxidase-active structure of cyt c. Subsequent self-oxidation eventually causes deactivation. It appears that this is the first report that identifies H2O2-induced covalent modifications as an essential component for the peroxidase activity of native cyt c

Calcium-Mediated Control of S100 Proteins: Allosteric Communication via an Agitator/Signal Blocking Mechanism.

Allosteric proteins possess dynamically coupled residues for the propagation of input signals to distant target binding sites. The input signals usually correspond to effector is present or effector is not present . Many aspects of allosteric regulation remain incompletely understood. This work focused on S100A11, a dimeric EF-hand protein with two hydrophobic target binding sites. An annexin peptide (Ax) served as the target. Target binding is allosterically controlled by Ca2+ over a distance of ∼26 Å. Ca2+ promotes formation of a [Ca4 S100 Ax2] complex, where the Ax peptides are accommodated between helices III/IV and III\u27/IV\u27. Without Ca2+ these binding sites are closed, precluding interactions with Ax. The allosteric mechanism was probed by microsecond MD simulations in explicit water, complemented by hydrogen exchange mass spectrometry (HDX/MS). Consistent with experimental data, MD runs in the absence of Ca2+ and Ax culminated in target binding site closure. In simulations on [Ca4 S100] the target binding sites remained open. These results capture the essence of allosteric control, revealing how Ca2+ prevents binding site closure. Both HDX/MS and MD data showed that the metalation sites become more dynamic after Ca2+ loss. However, these enhanced dynamics do not represent the primary trigger of the allosteric cascade. Instead, a labile salt bridge acts as an incessantly active agitator that destabilizes the packing of adjacent residues, causing a domino chain of events that culminates in target binding site closure. This agitator represents the starting point of the allosteric signal propagation pathway. Ca2+ binding rigidifies elements along this pathway, thereby blocking signal transmission. This blocking mechanism does not conform to the commonly held view that allosteric communication pathways generally originate at the sites where effectors interact with the protein

Impact of Autosomal Recessive Juvenile Parkinson’s Disease Mutations on the Structure and Interactions of the Parkin Ubiquitin-like Domain

Autosomal recessive juvenile parkinsonism (ARJP) is an early onset familial form of Parkinson\u27s disease. Approximately 50% of all ARJP cases are attributed to mutations in the gene park2, coding for the protein parkin. Parkin is a multidomain E3 ubiquitin ligase with six distinct domains including an N-terminal ubiquitin-like (Ubl) domain. In this work we examined the structure, stability, and interactions of the parkin Ubl domain containing most ARJP causative mutations. Using NMR spectroscopy we show that the Ubl domain proteins containing the ARJP substitutions G12R, D18N, K32T, R33Q, P37L, and K48A retained a similar three-dimensional fold as the Ubl domain, while at least one other (V15M) had altered packing. Four substitutions (A31D, R42P, A46P, and V56E) result in poor folding of the domain, while one protein (T55I) showed evidence of heterogeneity and aggregation. Further, of the substitutions that maintained their three-dimensional fold, we found that four of these (V15M, K32T, R33Q, and P37L) lead to impaired function due to decreased ability to interact with the 19S regulatory subunit S5a. Three substitutions (G12R, D18N, and Q34R) with an uncertain role in the disease did not alter the three-dimensional fold or S5a interaction. This work provides the first extensive characterization of the structural effects of causative mutations within the ubiquitin-like domain in ARJP

Unmasking the Annexin I Interaction from the Structure of Apo-S100A11

AbstractS100A11 is a homodimeric EF-hand calcium binding protein that undergoes a calcium-induced conformational change and interacts with the phospholipid binding protein annexin I to coordinate membrane association. In this work, the solution structure of apo-S100A11 has been determined by NMR spectroscopy to uncover the details of its calcium-induced structural change. Apo-S100A11 forms a tight globular structure having a near antiparallel orientation of helices III and IV in calcium binding site II. Further, helices I and IV, and I and I′, form a more closed arrangement than observed in other apo-S100 proteins. This helix arrangement in apo-S100A11 partially buries residues in helices I (P3, E11, A15), III (V55, R58, M59), and IV (A86, C87, S90) and the linker (A45, F46), which are required for interaction with annexin I in the calcium-bound state. In apo-S100A11, this results in a “masked” binding surface that prevents annexin I binding but is uncovered upon calcium binding

Orion\u27s Bar: Physical Conditions Across the Definitive H\u3csup\u3e+\u3c/sup\u3e/H\u3csup\u3e0\u3c/sup\u3e/H\u3csub\u3e2\u3c/sub\u3e Interface

Previous work has shown the Orion Bar to be an interface between ionized and molecular gas, viewed roughly edge-on, which is excited by the light from the Trapezium cluster. Much of the emission from any star-forming region will originate from such interfaces, so the Bar serves as a foundation test of any emission model. Here we combine X-ray, optical, infrared (IR), and radio data sets to derive emission spectra along the transition from H+ to H0 to H2 regions. We then reproduce the spectra of these layers with a simulation that simultaneously accounts for the detailed microphysics of the gas, the grains, and molecules, especially H2 and CO. The magnetic field, observed to be the dominant pressure in another region of the Orion Nebula, is treated as a free parameter, along with the density of cosmic rays. Our model successfully accounts for the optical, IR, and radio observations across the Bar by including a significant magnetic pressure and also heating by an excess density of cosmic rays, which we suggest is due to cosmic rays being trapped in the compressed magnetic field. In the Orion Bar, as we had previously found in M17, momentum carried by radiation and winds from the newly formed stars pushes back and compresses the surrounding gas. There is a rough balance between outward momentum in starlight and the total pressure in atomic and molecular gas surrounding the H+ region. If the gas starts out with a weak magnetic field, the starlight from a newly formed cluster will push back the gas and compress the gas, magnetic field, and cosmic rays until magnetic pressure becomes an important factor

Synergistic recruitment of UbcH7~Ub and phosphorylated Ubl domain triggers parkin activation

The E3 ligase parkin ubiquitinates outer mitochondrial membrane proteins during oxidative stress and is linked to early-onset Parkinson’s disease. Parkin is autoinhibited but is activated by the kinase PINK1 that phosphorylates ubiquitin leading to parkin recruitment, and stimulates phosphorylation of parkin’s N-terminal ubiquitin-like (pUbl) domain. How these events alter the structure of parkin to allow recruitment of an E2~Ub conjugate and enhanced ubiquitination is an unresolved question. We present a model of an E2~Ub conjugate bound to the phosphoubiquitin-loaded C-terminus of parkin, derived from NMR chemical shift perturbation experiments. We show the UbcH7~Ub conjugate binds in the open state whereby conjugated ubiquitin binds to the RING1/IBR interface. Further, NMR and mass spectrometry experiments indicate the RING0/RING2 interface is re-modelled, remote from the E2 binding site, and this alters the reactivity of the RING2(Rcat) catalytic cysteine, needed for ubiquitin transfer. Our experiments provide evidence that parkin phosphorylation and E2~Ub recruitment act synergistically to enhance a weak interaction of the pUbl domain with the RING0 domain and rearrange the location of the RING2(Rcat) domain to drive parkin activity

Structure of an Asymmetric Ternary Protein Complex Provides Insight for Membrane Interaction

SummaryPlasma membrane repair involves the coordinated effort of proteins and the inner phospholipid surface to mend the rupture and return the cell back to homeostasis. Here, we present the three-dimensional structure of a multiprotein complex that includes S100A10, annexin A2, and AHNAK, which along with dysferlin, functions in muscle and cardiac tissue repair. The 3.5 Å resolution X-ray structure shows that a single region from the AHNAK C terminus is recruited by an S100A10-annexin A2 heterotetramer, forming an asymmetric ternary complex. The AHNAK peptide adopts a coil conformation that arches across the heterotetramer contacting both annexin A2 and S100A10 protomers with tight affinity (∼30 nM) and establishing a structural rationale whereby both S100A10 and annexin proteins are needed in AHNAK recruitment. The structure evokes a model whereby AHNAK is targeted to the membrane surface through sandwiching of the binding region between the S100A10/annexin A2 complex and the phospholipid membrane

A Photometrically and Morphologically Variable Infrared Nebula in L483

We present narrow and broad K-band observations of the Class 0/I source IRAS 18148-0440 that span 17 years. The infrared nebula associated with this protostar in the L483 dark cloud is both morphologically and photometrically variable on a time scale of only a few months. This nebula appears to be an infrared analogue to other well-known optically visible variable nebulae associated with young stars, such as Hubble's Variable Nebula. Along with Cepheus A, this is one of the first large variable nebulae to be found that is only visible in the infrared. The variability of this nebula is most likely due to changing illumination of the cloud rather than any motion of the structure in the nebula. Both morphological and photometric changes are observed on a time scale only a few times longer than the light crossing time of the nebula, suggesting very rapid intrinsic changes in the illumination of the nebula. Our narrow-band observations also found that H_2 knots are found nearly twice as far to the east of the source as to its west, and that H_2 emission extends farther east of the source than the previously known CO outflow.Comment: 19 pages, 6 figures, 1 tabl

Conserved region 3 of human papillomavirus 16 E7 contributes to deregulation of the retinoblastoma tumor suppressor

The human papillomavirus (HPV) E7 oncoprotein binds cellular factors, preventing or retargeting their function and thereby making the infected cell conducive for viral replication. A key target of E7 is the product of the retinoblastoma susceptibility locus (pRb). This interaction results in the release of E2F transcription factors and drives the host cell into the S phase of the cell cycle. E7 binds pRb via a high-affinity binding site in conserved region 2 (CR2) and also targets a portion of cellular pRb for degradation via the proteasome. Evidence suggests that a secondary binding site exists in CR3, and that this interaction influences pRb deregulation. Additionally, evidence suggests that CR3 also participates in the degradation of pRb. We have systematically analyzed the molecular mechanisms by which CR3 contributes to deregulation of the pRb pathway by utilizing a comprehensive series of mutations in residues predicted to be exposed on the surface of HPV16 E7 CR3. Despite differences in the ability to interact with cullin 2, all CR3 mutants degrade pRb comparably to wild-type E7. We identified two specific patches of residues on the surface of CR3 that contribute to pRb binding independently of the high-affinity CR2 binding site. Mutants within CR3 that affect pRb binding are less effective than the wild-type E7 in overcoming pRb-induced cell cycle arrest. This demonstrates that the interaction between HPV16 E7 CR3 and pRb is functionally important for alteration of the cell cycle. © 2012, American Society for Microbiology