Checking the conformational stability of cystatin C and its L68Q variant by molecular dynamics studies: Why is the L68Q variant amyloidogenic?

Rodziewicz‐Motowidło, Sylwia; Wahlbom, Maria; Wang, Xin; Lagiewka, J; Janowski, Robert; Jaskólski, M.; Grubb, Anders; Grzonka, Zbigniew

doi:10.1016/j.jsb.2005.11.015

Cited by 26 publications

(44 citation statements)

References 50 publications

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“…Briefly, plasmid pET11A coding for dsGB1 was a gift from Angela Gronenborn (University of Pittsburgh). Uniformly and selectively 13 C-and/or 15 N-labeled protein was obtained by expression in Escherichia coli HMS174 (DE3), using M9 minimal medium and isotopically labeled materials from Cambridge Isotope Laboratories (Andover, MA) and Isotec (Sigma-Aldrich). 13 CO Tyr-labeled sample was prepared as previously described (37).…”

Section: Methodsmentioning

confidence: 99%

“…Amyloid fibrils of 15 N-labeled dsGB1 were centrifuged at 1 H frequencies of 600 and 800 MHz). Spinning rates were typically 13 kHz (at 14.1 T) or 17 kHz (at 18.8 T) unless stated otherwise, with sample cooling at 267 K. Standard two-dimensional and three-dimensional MAS ssNMR assignment experiments were employed to assign the resonances, mostly via NCOCX and NCACX experiments, along with two-dimensional 13 C-13 C spectra obtained with dipolar-assisted rotational resonance (39) and phase-alternated recoupling irradiation scheme (PARIS) (40) 13 C-13 C mixing. A series of two-dimensional 13 C-13 C NMR spectra with 23-, 54-, and 100-ms PARIS mixing were used to probe longer distance 13 C- 13 C contacts (at 17-kHz MAS and 800-MHz 1 H frequencies).…”

Section: Methodsmentioning

confidence: 99%

“…Spinning rates were typically 13 kHz (at 14.1 T) or 17 kHz (at 18.8 T) unless stated otherwise, with sample cooling at 267 K. Standard two-dimensional and three-dimensional MAS ssNMR assignment experiments were employed to assign the resonances, mostly via NCOCX and NCACX experiments, along with two-dimensional 13 C-13 C spectra obtained with dipolar-assisted rotational resonance (39) and phase-alternated recoupling irradiation scheme (PARIS) (40) 13 C-13 C mixing. A series of two-dimensional 13 C-13 C NMR spectra with 23-, 54-, and 100-ms PARIS mixing were used to probe longer distance 13 C- 13 C contacts (at 17-kHz MAS and 800-MHz 1 H frequencies). To probe the inter-strand register, PITHIRDS-CT measurements (41,42) were carried out using fibrils prepared from Tyr-1-13 CO-labeled dsGB1 at 600 MHz ( 1 H frequency) and an MAS frequency of 20 kHz, with up to 38.4 ms of total dipolar recoupling.…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

“…The amyloid-forming protein cystatin C, which is the cause of a cerebral amyloidosis, is a key example. Certain mutants form amyloid fibrils and also undergo domain swapping, and inhibition of domain swapping also affects fibril formation (13)(14)(15)(16)(17). Domain swapping has also been proposed as an oligomeriza-tion mechanism for transthyretin (18), ␤2m (19 -21), and the prion protein (22,23), although arguably with less direct evidence to support these cases.…”

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confidence: 99%

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Amyloid-like Fibrils from a Domain-swapping Protein Feature a Parallel, in-Register Conformation without Native-like Interactions

Hoop

Kodali

et al. 2011

Journal of Biological Chemistry

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The formation of amyloid-like fibrils is characteristic of various diseases, but the underlying mechanism and the factors that determine whether, when, and how proteins form amyloid, remain uncertain. Certain mechanisms have been proposed based on the three-dimensional or runaway domain swapping, inspired by the fact that some proteins show an apparent correlation between the ability to form domain-swapped dimers and a tendency to form fibrillar aggregates. Intramolecular ␤-sheet contacts present in the monomeric state could constitute intermolecular ␤-sheets in the dimeric and fibrillar states. One example is an amyloid-forming mutant of the immunoglobulin binding domain B1 of streptococcal protein G, which in its native conformation consists of a four-stranded ␤-sheet and one ␣-helix. Under native conditions this mutant adopts a domainswapped dimer, and it also forms amyloid-like fibrils, seemingly in correlation to its domain-swapping ability. We employ magic angle spinning solid-state NMR and other methods to examine key structural features of these fibrils. Our results reveal a highly rigid fibril structure that lacks mobile domains and indicate a parallel in-register ␤-sheet structure and a general loss of native conformation within the mature fibrils. This observation contrasts with predictions that native structure, and in particular intermolecular ␤-strand interactions seen in the dimeric state, may be preserved in "domain-swapping" fibrils. We discuss these observations in light of recent work on related amyloidforming proteins that have been argued to follow similar mechanisms and how this may have implications for the role of domain-swapping propensities for amyloid formation.Amyloid fibril formation is characteristic of a variety of human disorders, including Huntington and Alzheimer diseases (1, 2). In amyloid-related diseases, one or more proteins are found in fibrillar aggregates, in a non-native, highly ␤-sheetrich conformation. Depending on the disease context, the propensity for amyloid formation may be traced to mutations and cleavage events, combined with poorly understood external triggers. Many proteins can also be made to form amyloid fibrils in vitro. Intriguingly, there are many accounts of proteins that form amyloid-like fibrils that are not associated with pathologies (2). Whether disease-related or not, amyloid fibrils share key biochemical and biophysical characteristics, suggesting common structural features. Understanding the fibril formation pathway is of interest not only as there may be a correlation between disease onset and protein aggregation, but also because transient oligomeric precursors may act as toxic species. However, a lack of high resolution structures of most fibrils and their precursors limits our knowledge of the mechanism of formation.One seemingly common structural motif for amyloid fibrils is an in-register parallel (IP) 3 assembly into pleated ␤-sheets, stabilized by backbone-to-backbone hydrogen bonding, combined with favorable side-chain interactions (e....

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Amyloid-like Fibrils from a Domain-swapping Protein Feature a Parallel, in-Register Conformation without Native-like Interactions

Hoop

Kodali

et al. 2011

Journal of Biological Chemistry

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The role of the Val57 amino‐acid residue in the hinge loop of the human cystatin C. Conformational studies of the beta2‐L1‐beta3 segments of wild‐type human cystatin C and its mutants

et al. 2009

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Human cystatin C (HCC) is one of the amyloidogenic proteins to be shown to oligomerize via a three-dimensional domain swapping mechanism. This process precedes the formation of a stable dimer and proceeds particularly easily in the case of the L68Q mutant. According to the proposed mechanism, dimerization of the HCC precedes conformational changes within the beta2 and beta3 strands. In this article, we present conformational studies, using circular dichroism and MD methods, of the beta2-L1-beta3 (His43-Thr72) fragment of the HCC involved in HCC dimer formation. We also carried out studies of the beta2-L1-beta3 peptide, in which the Val57 residue was replaced by residues promoting beta-turn structure formation (Asp, Asn, or Pro). The present study established that point mutation could modify the structure of the L1 loop in the beta-hairpin peptide. Our results showed that the L1 loop in the peptide excised from human cystatin C is broader than that in cystatin C. In the HCC protein, broadening of the L1 loop together with the unfavorable L68Q mutation in the hydrophobic pocket could be a force sufficient to cause the partial unfolding and then the opening of HCC or its L68Q mutant structure for further dimerization. We presume further that the Asp57 and Asn57 mutations in the L1 loop of HCC could stabilize the closed form of HCC, whereas the Pro57 mutation could lead to the opening of the HCC structure and then to dimer/oligomer formation.

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Identification of the epitope for anti‐cystatin C antibody

Sladewska

Szymańska

Kordalska

et al. 2011

J of Molecular Recognition

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Human cystatin C (hCC), like many other amyloidogenic proteins, has been shown to form dimers by exchange of subdomains of the monomeric protein. Considering the model of hCC fibrillogenesis by propagated domain swapping, it seems possible that inhibition of this process should also suppress the entire process of dimerization and fibrillogenesis which leads to specific amyloidosis (hereditary cystatin C amyloid angiopathy (HCCAA)). It was reported that exogenous agents like monoclonal antibody against cystatin C are able to suppress formation of cystatin C dimers. In the effort to find a way of controlling the cystatin fibrillization process, the interactions between monoclonal antibody Cyst-13 and cystatin C were studied in detail. The present work describes the determination of the epitope of hCC to a monoclonal antibody raised against cystatin C, Cyst-13, by MALDI mass spectrometry, using proteolytic excision of the immune complex. The shortest epitope sequence was determined as hCC(107-114). Affinity studies of synthetic peptides revealed that the octapeptide with epitope sequence does not have binding ability to Cyst-13, whereas its longer counterpart, hCC(105-114), binds the studied antibody. The secondary structure of the peptides with epitope sequence was studied using circular dichroism and NMR spectroscopy.

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Checking the conformational stability of cystatin C and its L68Q variant by molecular dynamics studies: Why is the L68Q variant amyloidogenic?

Cited by 26 publications

References 50 publications

Amyloid-like Fibrils from a Domain-swapping Protein Feature a Parallel, in-Register Conformation without Native-like Interactions

Amyloid-like Fibrils from a Domain-swapping Protein Feature a Parallel, in-Register Conformation without Native-like Interactions

The role of the Val57 amino‐acid residue in the hinge loop of the human cystatin C. Conformational studies of the beta2‐L1‐beta3 segments of wild‐type human cystatin C and its mutants

Identification of the epitope for anti‐cystatin C antibody

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