scholarly journals Structural Characterization of Covalently Stabilized Human Cystatin C Oligomers

2020 ◽  
Vol 21 (16) ◽  
pp. 5860
Author(s):  
Magdalena Chrabąszczewska ◽  
Adam K. Sieradzan ◽  
Sylwia Rodziewicz-Motowidło ◽  
Anders Grubb ◽  
Christopher M. Dobson ◽  
...  
Keyword(s):  
Cystatin C ◽  
Protein Misfolding ◽  
Structural Information ◽  
Outer Edge ◽  
Cysteine Protease Inhibitor ◽  
X Ray ◽  
X Ray Crystallography ◽  
Human Cystatin C ◽  
Dynamics Simulations ◽  
Human Cystatin

Human cystatin C (HCC), a cysteine-protease inhibitor, exists as a folded monomer under physiological conditions but has the ability to self-assemble via domain swapping into multimeric states, including oligomers with a doughnut-like structure. The structure of the monomeric HCC has been solved by X-ray crystallography, and a covalently linked version of HCC (stab-1 HCC) is able to form stable oligomeric species containing 10–12 monomeric subunits. We have performed molecular modeling, and in conjunction with experimental parameters obtained from atomic force microscopy (AFM), transmission electron microscopy (TEM) and small-angle X-ray scattering (SAXS) measurements, we observe that the structures are essentially flat, with a height of about 2 nm, and the distance between the outer edge of the ring and the edge of the central cavity is ~5.1 nm. These dimensions correspond to the height and diameter of one stab-1 HCC subunit and we present a dodecamer model for stabilized cystatin C oligomers using molecular dynamics simulations and experimentally measured parameters. Given that oligomeric species in protein aggregation reactions are often transient and very highly heterogeneous, the structural information presented here on these isolated stab-1 HCC oligomers may be useful to further explore the physiological relevance of different structural species of cystatin C in relation to protein misfolding disease.

scholarly journals Structural characterization of covalently stabilized human cystatin C oligomers

2019 ◽  
Author(s):  
Magdalena Chrabąszczewska ◽  
Adam K. Sieradzan ◽  
Sylwia Rodziewicz-Motowidło ◽  
Anders Grubb ◽  
Christopher M. Dobson ◽  
...  
Keyword(s):  
Cystatin C ◽  
Protein Misfolding ◽  
Structural Information ◽  
Outer Edge ◽  
Cysteine Protease Inhibitor ◽  
X Ray Crystallography ◽  
Human Cystatin C ◽  
Covalently Linked ◽  
Dynamics Simulations ◽  
Human Cystatin

AbstractHuman cystatin C (HCC), a cysteine-protease inhibitor, exists as a folded monomer under physiological conditions but has the ability to self-assemble via domain swapping into multimeric states, including oligomers with a doughnut-like structure. The structure of the monomeric HCC has been solved by X-ray crystallography, and a covalently linked version of HCC (stab-1 HCC) is able to form stable oligomeric species containing 10-12 monomeric subunits. We have performed molecular modeling, and in conjunction with experimental parameters obtained from AFM, TEM and SAXS measurements, we observe that the structures are essentially flat, with a height of about 2 nm, and the distance between the outer edge of the ring and the edge of the central cavity is ~5.1 nm. These dimensions correspond to the height and diameter of one stab-1 HCC subunit and we present a dodecamer model for stabilized cystatin C oligomers using molecular dynamics simulations and experimentally measured parameters. Given that oligomeric species in protein aggregation reactions are often transient and very highly heterogeneous, the structural information presented here on these isolated stab-1 HCC oligomers may provide useful to further explore the physiological relevance of different structural species of cystatin C in relationship to protein misfolding disease

scholarly journals Revealing a hidden intermediate of rotatory catalysis with X-ray crystallography and Molecular simulations

2021 ◽  
Author(s):  
Mrinal Shekhar ◽  
Chitrak Gupta ◽  
Kano Suzuki ◽  
Abhishek Singharoy ◽  
Takeshi Murata
Keyword(s):  
Molecular Motors ◽  
Atp Hydrolysis ◽  
Structural Information ◽  
Hydrolysis Product ◽  
X Ray ◽  
X Ray Crystallography ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Dynamics Simulations ◽  
Rate Limiting

The mechanism of rotatory catalysis in ATP-hydrolyzing molecular motors remain an unresolved puzzle in biological energy transfer. Notwithstanding the wealth of available biochemical and structural information inferred from years of experiments, knowledge on how the coupling between the chemical and mechanical steps within motors enforces directional rotatory movements remains fragmentary. Even more contentious is to pinpoint the rate-limiting step of a multi-step rotation process. Here, using Vacuolar or V1-type hexameric ATPase as an exemplary rotational motor, we present a model of the complete 4-step conformational cycle involved in rotatory catalysis. First, using X-ray crystallography a new intermediate or 'dwell' is identified, which enables the release of an inorganic phosphate (or Pi) after ATP hydrolysis. Using molecular dynamics simulations, this new dwell is placed in a sequence with three other crystal structures to derive a putative cyclic rotation path. Free-energy simulations are employed to estimate the rate of the hexameric protein transfor-mations, and delineate allosteric effects that allow new reactant ATP entry only after hydrolysis product exit. An analysis of transfer entropy brings to light how the sidechain level interactions transcend into larger scale reorganizations, highlighting the role of the ubiquitous arginine-finger residues in coupling chemical and mechanical information. Inspection of all known rates encompassing the 4-step rotation mechanism implicates overcoming of the ADP interactions with V1-ATPase to be the rate-limiting step of motor action.

scholarly journals Expression of a selenomethionyl derivative and preliminary crystallographic studies of human cystatin C

1999 ◽  
Vol 55 (11) ◽  
pp. 1939-1942 ◽  
Author(s):  
Maciej Kozak ◽  
Elzbieta Jankowska ◽  
Robert Janowski ◽  
Zbigniew Grzonka ◽  
Anders Grubb ◽  
...  
Keyword(s):  
Cystatin C ◽  
Potent Inhibitor ◽  
Full Length ◽  
X Ray ◽  
Cubic Form ◽  
Human Cystatin C ◽  
Multiple Copies ◽  
Polymorphic Forms ◽  
X Ray Absorption ◽  
Human Cystatin

Human cystatin C, a protein with amyloidogenic properties and a potent inhibitor of papain-like mammalian proteases, has been produced in its full-length form by recombinant techniques and crystallized in two polymorphic forms: cubic and tetragonal. A selenomethionyl derivative of the protein, obtained by Escherichia coli expression and with complete Met→Se-Met substitution confirmed by mass spectrometry, amino-acid analysis and X-ray absorption spectra, was crystallized in the cubic form. A truncated variant of the protein, lacking ten N-terminal residues, has also been crystallized. The crystals of this variant are tetragonal and, like the two polymorphs of the full-length protein, contain multiple copies of the molecule in the asymmetric unit, suggesting oligomerization of the protein.

Molecular Dynamics Simulations To Investigate the Domain Swapping Mechanism of Human Cystatin C

2008 ◽  
Vol 23 (3) ◽  
pp. 577-584 ◽  
Author(s):  
Yuan-Min Lin ◽  
Hsuan-Liang Liu ◽  
Jian-Hua Zhao ◽  
Chi-Hung Huang ◽  
Hsu-Wei Fang ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Molecular Dynamics Simulations ◽  
Cystatin C ◽  
Domain Swapping ◽  
Human Cystatin C ◽  
Dynamics Simulations ◽  
Human Cystatin

scholarly journals DMPC Phospholipid Bilayer as a Potential Interface for Human Cystatin C Oligomerization: Analysis of Protein-Liposome Interactions Using NMR Spectroscopy

Membranes ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 13
Author(s):  
Przemyslaw Jurczak ◽  
Kosma Szutkowski ◽  
Slawomir Lach ◽  
Stefan Jurga ◽  
Paulina Czaplewska ◽  
...  
Keyword(s):  
Cystatin C ◽  
Phospholipid Bilayer ◽  
Size Exclusion ◽  
Cysteine Protease Inhibitor ◽  
Vast Number ◽  
Membrane Mimetic ◽  
Human Cystatin C ◽  
Exclusion Chromatography ◽  
Cellular Components ◽  
Human Cystatin

Studies revolving around mechanisms responsible for the development of amyloid-based diseases lay the foundations for the recognition of molecular targets of future to-be-developed treatments. However, the vast number of peptides and proteins known to be responsible for fibril formation, combined with their complexity and complexity of their interactions with various cellular components, renders this task extremely difficult and time-consuming. One of these proteins, human cystatin C (hCC), is a well-known and studied cysteine-protease inhibitor. While being a monomer in physiological conditions, under the necessary stimulus—usually a mutation, it tends to form fibrils, which later participate in the disease development. This process can potentially be regulated (in several ways) by many cellular components and it is being hypothesized that the cell membrane might play a key role in the oligomerization pathway. Studies involving cell membranes pose several difficulties; therefore, an alternative in the form of membrane mimetics is a very attractive solution. Here, we would like to present the first study on hCC oligomerization under the influence of phospholipid liposomes, acting as a membrane mimetic. The protein–mimetic interactions are studied utilizing circular dichroism, nuclear magnetic resonance, and size exclusion chromatography.

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