Modeling studies of the change in conformation required for cleavage of limited proteolytic sites

Hubbard, Simon J.; Eisenmenger, Frank; Thornton, Janet M.

doi:10.1002/pro.5560030505

Cited by 215 publications

(221 citation statements)

References 47 publications

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“…Because each cleavage site must necessarily be (i) solvent-exposed and (ii) unstructured, we assign a repulsive interaction of energy E 0 between the cleavage site j and every other residue in the system. Because residues near a solvent-exposed site are also likely to be solvent-exposed, and because an unstructured region of ∼12 residues surrounding the cleavage site is necessary for the proteolytic enzyme to gain access to the site (27,44), we apply a stepwise, decreasingly repulsive potential to the two residues on either side of each cleavage site, such that the pairwise repulsive interaction of the respective residue with each residue in the system has energy:…”

Section: Methodsmentioning

confidence: 99%

Nonnative SOD1 trimer is toxic to motor neurons in a model of amyotrophic lateral sclerosis

Proctor

Fee

Tao

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

108

146

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Since the linking of mutations in the Cu,Zn superoxide dismutase gene (sod1) to amyotrophic lateral sclerosis (ALS) in 1993, researchers have sought the connection between SOD1 and motor neuron death. Disease-linked mutations tend to destabilize the native dimeric structure of SOD1, and plaques containing misfolded and aggregated SOD1 have been found in the motor neurons of patients with ALS. Despite advances in understanding of ALS disease progression and SOD1 folding and stability, cytotoxic species and mechanisms remain unknown, greatly impeding the search for and design of therapeutic interventions. Here, we definitively link cytotoxicity associated with SOD1 aggregation in ALS to a nonnative trimeric SOD1 species. We develop methodology for the incorporation of low-resolution experimental data into simulations toward the structural modeling of metastable, multidomain aggregation intermediates. We apply this methodology to derive the structure of a SOD1 trimer, which we validate in vitro and in hybridized motor neurons. We show that SOD1 mutants designed to promote trimerization increase cell death. Further, we demonstrate that the cytotoxicity of the designed mutants correlates with trimer stability, providing a direct link between the presence of misfolded oligomers and neuron death. Identification of cytotoxic species is the first and critical step in elucidating the molecular etiology of ALS, and the ability to manipulate formation of these species will provide an avenue for the development of future therapeutic strategies.neurodegeneration | protein aggregation | protein misfolding | ALS | structural modeling P rotein misfolding and aggregation are linked to cell death and disease progression in neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS). In these diseases and others, the formation of amyloid plaques, often observed post mortem, has long been thought to play a role in neurodegeneration, but toxicity has never been confirmed (1-3). Recent research has shown that small, soluble oligomers, rather than insoluble amyloids, are likely to be the cytotoxic species causing neurodegeneration (4-14). These small, soluble oligomers undergo aberrant interactions with cell machinery and activate cell death pathways, but their exact stoichiometry is not known and their properties have yet to be characterized. Recently, metastable soluble Cu,Zn superoxide dismutase (SOD1) oligomers have been identified that contain an epitope associated with disease-linked species of SOD1, mutants of which are implicated in a subset of ALS (15-18). Size exclusion chromatography (SEC) of these oligomers revealed a size range of two to four monomers, consistent with previous findings of potentially cytotoxic SOD1 oligomers (19)(20)(21).Knowledge of the structures of these species would not only allow for definitive testing of their toxicity but could potentially lead to an understanding of disease mechanism and therapeutic strategies against diseases for which no ...

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Section: Methodsmentioning

confidence: 99%

Nonnative SOD1 trimer is toxic to motor neurons in a model of amyotrophic lateral sclerosis

Proctor

Fee

Tao

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

108

146

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“…S these peptide bonds in the RNase molecule dissolved in 30-50% TFE are embedded in a rigid protein structure and thus not amenable to proteolytic digestion. Accepting our view that segmental mobility is the most critical parameter in dictating selective proteolysis of a globular protein (Fontana et al, 1986(Fontana et al, , 1993, it can be proposed that, with RNase in aqueous TFE, only the chain segment encompassing the site(s) of cleavage is flexible enough to bind easily and properly at the active site of thermolysin in order to facilitate peptide bond fission (Hubbard et al, 1994) (see also Polverino de Laureto et al, 1995b. for additional comments on the mechanism of selective proteolysis of proteins by thermolysin in aqueous TFE).…”

Section: Limited Proteolysismentioning

confidence: 99%

“…Indeed, a clear-cut correlation exists between sites of limited proteolysis and sites of high segmental mobility (B-factor) determined crystallographically (Fontana et al, 1986). It is our belief that notions of exposure, accessibility, or protrusion (Novotny & Bruccoleri, 1987;Hubbard et al, 1991) are clearly not sufficient to explain the limited proteolysis phenomenon, because it is evident that, even in a small globular protein, there are many exposed sites (see also Hubbard et al, 1994).…”

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confidence: 99%

Limited proteolysis of ribonuclease A with thermolysin in trifluoroethanol

et al. 1997

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We have examined the proteolysis of bovine pancreatic ribonuclease A (RNase) by thermolysin when dissolved in aqueous buffer, pH 7.0, in the presence of 50% (v/v) trifluoroethanol (TFE). Under these solvent conditions, RNase acquires a conformational state characterized by an enhanced content of secondary structure (helix) and reduced tertiary structure, as given by CD measurements. It was found that the TFE-resistant thermolysin, despite its broad substrate specificity, selectively cleaves the 124-residue chain of RNase in its TFE state (20-42"C, 6-24 h) at peptide bond Asn 34-Leu 35, followed by a slower cleavage at peptide bond Thr 45-Phe 46. In the absence of TFE, native RNase is resistant to proteolysis by thermolysin. Two nicked RNase species, resulting from cleavages at one or two peptide bonds and thus constituted by two (1-34 and 35-124) (RNase Thl) or three (1-34, 35-45 and 46-124) (RNase Th2) fragments linked covalently by the four disulfide bonds of the protein, were isolated to homogeneity by chromatography and characterized. CD measurements provided evidence that RNase Thl maintains the overall conformational features of the native protein, but shows a reduced thermal stability with respect to that of the intact species (-ATm 16 "C); RNase Th2 instead is fully unfolded at room temperature. That the structure of RNase Thl is closely similar to that of the intact protein was confirmed unambiguously by two-dimensional NMR measurements. Structural differences between the two protein species are located only at the level of the chain segment 30-41, i.e., at residues nearby the cleaved Asn 34-Leu 35 peptide bond. RNase Thl retained about 20% of the catalytic activity of the native enzyme, whereas RNase Th2 was inactive. The 3 1-39 segment of the polypeptide chain in native RNase forms an exposed and highly flexible loop, whereas the 41-48 region forms a &strand secondary structure containing active site residues. Thus, the conformational, stability, and functional properties of nicked RNase Thl and Th2 are in line with the concept that proteins appear to tolerate extensive structural variations only at their flexible or loose parts exposed to solvent. We discuss the conformational features of RNase in its TFE-state that likely dictate the selective proteolysis phenomenon by thermolysin.Keywords: CD; limited proteolysis; NMR; ribonuclease A; thermolysin; trifluoroethanol Short-chain alcohols, such as methanol, ethanol, propanol, chloroethanol, and, in particular, trifluoroethanol, have been used frequently to induce a-helical conformations in otherwise randomly coiled or partially structured polypeptides (Toniolo et al., 1975;

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“…20,[25][26][27][28][29] These methods include NMR spectroscopy; 20,26,[30][31][32] missing electron density in X-ray crystallography maps; 33 optical rotatory dispersion spectroscopy (ORD); 18,34 circular dichroism spectroscopy in the near-UV 35 and far-UV regions; 18,34,36,37 Raman spectroscopy and Raman optical activity; 38 Fourier transform infrared spectroscopy (FTIR); 18 gelfiltration, viscometry, small angle neutron scattering (SANS), small angle X-ray scattering (SAXS), sedimentation, and dynamic and static light scattering; 27,39,40 fluorescent spectroscopy; 27,40 aberrant mobility in SDS-gel electrophoresis; 41,42 limited proteolysis (including conventional limited proteolysis [43][44][45][46][47] pulse proteolysis, 48 limited proteolysis combined to combined mass spectrometry, 49 and rapid and simple thermal proteolysis FASTpp assays; 50 H/D exchange; 27 abnormal conformational stability;. 40,[51][52][53][54] immunochemical methods; 55,…”

Section: Introductionmentioning

confidence: 99%

How disordered is my protein and what is its disorder for? A guide through the “dark side” of the protein universe

Lieutaud

Ferrón

Uversky

et al. 2016

Intrinsically Disordered Proteins

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In the last 2 decades it has become increasingly evident that a large number of proteins are either fully or partially disordered. Intrinsically disordered proteins lack a stable 3D structure, are ubiquitous and fulfill essential biological functions. Their conformational heterogeneity is encoded in their amino acid sequences, thereby allowing intrinsically disordered proteins or regions to be recognized based on properties of these sequences. The identification of disordered regions facilitates the functional annotation of proteins and is instrumental for delineating boundaries of protein domains amenable to structural determination with X-ray crystallization. This article discusses a comprehensive selection of databases and methods currently employed to disseminate experimental and putative annotations of disorder, predict disorder and identify regions involved in induced folding. It also provides a set of detailed instructions that should be followed to perform computational analysis of disorder.KEYWORDS disorder databases and metaservers; induced folding; intrinsic disorder; intrinsically disordered proteins; intrinsically disordered regions; prediction methods

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Modeling studies of the change in conformation required for cleavage of limited proteolytic sites

Cited by 215 publications

References 47 publications

Nonnative SOD1 trimer is toxic to motor neurons in a model of amyotrophic lateral sclerosis

Nonnative SOD1 trimer is toxic to motor neurons in a model of amyotrophic lateral sclerosis

Limited proteolysis of ribonuclease A with thermolysin in trifluoroethanol

How disordered is my protein and what is its disorder for? A guide through the “dark side” of the protein universe

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