Structure of the Calcium Pump from Sarcoplasmic Reticulum at 8 Å Resolution: Architecture of the Transmembrane Helices and Localization of the Binding Site for Thapsigargin

1998 ◽  
Vol 4 (S2) ◽  
pp. 462-463
Author(s):  
P. Zhang ◽  
C. Toyoshima ◽  
K. Yonekura ◽  
G. Inesi ◽  
M. Green ◽  
...  
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Transmembrane Domain ◽  
Three Dimensional ◽  
Specific Inhibitor ◽  
Large Family ◽  
Calcium Pump ◽  
Dimensional Structure ◽  
Transmembrane Helices ◽  
Site Mutagenesis ◽  
P Type

The calcium pump (Ca2+-ATPase) from sarcoplasmic reticulum (SR) is a prominent member of the large family of ATP-dependent cation pumps, which include Na+ /K+-ATPase, H+/K+-ATPase from the stomach, H+-ATPase from yeast and Neurospora, and various detoxifying pumps for Cd+, Cu+ and other metals. In muscle, calcium is stored inside the SR and contraction is initiated by regulated release through specific calcium channels; Ca2+ -ATPase is responsible for relaxation by pumping calcium back into the SR lumen. Many techniques (chemical modification, site mutagenesis, reaction kinetics) have been used to correlate Ca2+-ATPase sequence with function, but no high resolution three-dimensional structure of Ca2+-ATPase, or any P-type pump, has yet been determined. In the current work, we have determined the structure from helical crystals at 8 A resolution and thus revealed the alpha-helical architecture of the transmembrane domain. In addition, a specific inhibitor of Ca2+-ATPase, thapsigargin, was used to promote crystallization and we have characterized the structural consequences of its inhibition.

scholarly journals Relationship between sequence conservation and three-dimensional structure in a large family of esterases, lipases, and related proteins

1993 ◽  
Vol 2 (3) ◽  
pp. 366-382 ◽  
Author(s):  
Miroslaw Cygler ◽  
Joseph D. Schrag ◽  
Joel L. Sussman ◽  
Michal Harel ◽  
Israel Silman ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Large Family ◽  
Sequence Conservation ◽  
Dimensional Structure ◽  
Three Dimensional Structure ◽  
Related Proteins

scholarly journals Topography Prediction of Helical Transmembrane Proteins by a New Modification of the Sliding Window Method

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Maria N. Simakova ◽  
Nikolai N. Simakov
Keyword(s):  
Membrane Proteins ◽  
Transmembrane Domain ◽  
Domain Boundary ◽  
Three Dimensional ◽  
Transmembrane Proteins ◽  
Sliding Window ◽  
Dimensional Structure ◽  
X Ray ◽  
X Ray Crystallography ◽  
Window Method

Protein functions are specified by its three-dimensional structure, which is usually obtained by X-ray crystallography. Due to difficulty of handling membrane proteins experimentally to date the structure has only been determined for a very limited part of membrane proteins (<4%). Nevertheless, investigation of structure and functions of membrane proteins is important for medicine and pharmacology and, therefore, is of significant interest. Methods of computer modeling based on the data on the primary protein structure or the symbolic amino acid sequence have become an actual alternative to the experimental method of X-ray crystallography for investigating the structure of membrane proteins. Here we presented the results of the study of 35 transmembrane proteins, mainly GPCRs, using the novel method of cascade averaging of hydrophobicity function within the limits of a sliding window. The proposed method allowed revealing 139 transmembrane domains out of 140 (or 99.3%) identified by other methods. Also 236 transmembrane domain boundary positions out of 280 (or 84%) were predicted correctly by the proposed method with deviation from the predictions made by other methods that does not exceed the detection error of this method.

scholarly journals Structure and Functional Analysis of a Ribosomal Oxygenase

2014 ◽  
Vol 70 (a1) ◽  
pp. C1408-C1408
Author(s):  
Laura van Staalduinen ◽  
Stefanie Novakowski ◽  
Zongchao Jia
Keyword(s):  
Ribosomal Protein ◽  
Active Site ◽  
Three Dimensional ◽  
Large Family ◽  
Structural Similarity ◽  
Dimensional Structure ◽  
Titration Calorimetry ◽  
Ribosome Assembly ◽  
Oxidation Reactions ◽  
Jmjc Domain

The 2-oxoglutarate/Fe(II)-dependent oxygenases (2OG oxygenases) are a large family of proteins that share a similar overall three-dimensional structure and catalyze a diverse array of oxidation reactions. The Jumonji C (JmjC)-domain containing proteins represent an important subclass of the 2OG oxygenase family that typically catalyze protein hydroxylation; however, recently other reactions have been identified, such as tRNA modification. The E. coli gene, ycfD, was predicted to be a JmjC-domain containing protein of unknown function based on primary sequence. Recently YcfD was determined to act as a ribosomal oxygenase, hydroxylating an arginine residue on the 50S ribosomal protein L-16 (RL-16). We have determined the crystal structure of YcfD at 2.7 Å resolution, revealing that YcfD is structurally similar to known JmjC proteins and possesses the characteristic double stranded β-helix fold or cupin domain. Separate from the cupin domain, an additional globular module termed -helical arm mediates dimerization of YcfD. We further have shown that 2-oxoglutarate binds to YcfD using isothermal titration calorimetry and identified R140 and S116 as key 2OG binding residues using mutagenesis which, together with the iron location and structural similarity with other cupin family members, allowed identification of the active site. Structural homology to ribosomal assembly proteins combined with GST-YcfD pull-down of a ribosomal protein and docking of RL-16 to the YcfD active site support the role of YcfD in regulation of bacterial ribosome assembly. Furthermore, overexpression of YcfD is shown to inhibit cell growth signifying a toxic effect on ribosome assembly.

scholarly journals Biochemical Characterization of the Amylase Activity from the New Haloarchaeal Strain Haloarcula sp. HS Isolated in the Odiel Marshlands

Biology ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 337
Author(s):  
Patricia Gómez-Villegas ◽  
Javier Vigara ◽  
Luis Romero ◽  
Cecilia Gotor ◽  
Sara Raposo ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Amylase Activity ◽  
Extreme Environments ◽  
Three Dimensional ◽  
Large Family ◽  
Industrial Applications ◽  
Dimensional Structure ◽  
Glycosyl Hydrolases ◽  
Calcium Dependent ◽  
Alpha Amylases

Alpha-amylases are a large family of α,1-4-endo-glycosyl hydrolases distributed in all kingdoms of life. The need for poly-extremotolerant amylases encouraged their search in extreme environments, where archaea become ideal candidates to provide new enzymes that are able to work in the harsh conditions demanded in many industrial applications. In this study, a collection of haloarchaea isolated from Odiel saltern ponds in the southwest of Spain was screened for their amylase activity. The strain that exhibited the highest activity was selected and identified as Haloarcula sp. HS. We demonstrated the existence in both, cellular and extracellular extracts of the new strain, of functional α-amylase activities, which showed to be moderately thermotolerant (optimum around 60 °C), extremely halotolerant (optimum over 25% NaCl), and calcium-dependent. The tryptic digestion followed by HPLC-MS/MS analysis of the partially purified cellular and extracellular extracts allowed to identify the sequence of three alpha-amylases, which despite sharing a low sequence identity, exhibited high three-dimensional structure homology, conserving the typical domains and most of the key consensus residues of α-amylases. Moreover, we proved the potential of the extracellular α-amylase from Haloarcula sp. HS to treat bakery wastes under high salinity conditions.

scholarly journals Structure–Function Relationships of the Repeat Domains of RTX Toxins

Toxins ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 657 ◽  
Author(s):  
Ulrich Baumann
Keyword(s):  
Calcium Binding ◽  
Tandem Repeats ◽  
Three Dimensional ◽  
Large Family ◽  
Variable Number ◽  
General Purpose ◽  
Dimensional Structure ◽  
Type I ◽  
Structural Determinants ◽  
Extracellular Medium

RTX proteins are a large family of polypeptides of mainly Gram-negative origin that are secreted into the extracellular medium by a type I secretion system featuring a non-cleavable C-terminal secretion signal, which is preceded by a variable number of nine-residue tandem repeats. The three-dimensional structure forms a parallel β-roll, where β-strands of two parallel sheets are connected by calcium-binding linkers in such a way that a right-handed spiral is built. The Ca2+ ions are an integral part of the structure, which cannot form without them. The structural determinants of this unique architecture will be reviewed with its conservations and variations together with the implication for secretion and folding of these proteins. The general purpose of the RTX domains appears to act as an internal chaperone that keeps the polypeptide unfolded in the calcium-deprived cytosol and triggers folding in the calcium-rich extracellular medium. A rather recent addition to the structural biology of the RTX toxin is a variant occurring in a large RTX adhesin, where this non-canonical β-roll binds to ice and diatoms.

scholarly journals Identification of the Mg2+-binding site in the P-type ATPase and phosphatase members of the HAD (haloacid dehalogenase) superfamily by structural similarity to the response regulator protein CheY

1999 ◽  
Vol 339 (2) ◽  
pp. 223-226 ◽  
Author(s):  
Ivo S. RIDDER ◽  
Bauke W. DIJKSTRA
Keyword(s):  
Binding Site ◽  
Response Regulator ◽  
Three Dimensional ◽  
Structural Similarity ◽  
Dimensional Structure ◽  
Phosphate Binding ◽  
Haloacid Dehalogenase ◽  
Regulator Protein ◽  
P Type ◽  
Response Regulator Protein

The large HAD (haloacid dehalogenase) superfamily of hydrolases comprises P-type ATPases, phosphatases, epoxide hydrolases and l-2-haloacid dehalogenases. A comparison of the three-dimensional structure of l-2-haloacid dehalogenase with that of the response regulator protein CheY allowed the assignment of a conserved pair of aspartate residues as the Mg2+-binding site in the P-type ATPase and phosphatase members of the superfamily. From the resulting model of the active site, a conserved serine/threonine residue is suggested to be involved in phosphate binding, and a mechanism comprising a phosphoaspartate intermediate is postulated.

scholarly journals Structure and function of one unclassified-class glutathione S-transferase in Leptinotarsa decemlineata

2021 ◽  
Author(s):  
Yanjun Liu ◽  
Timothy W Moural ◽  
Sonu BK Koirala ◽  
Jonathan Hernandez ◽  
Zhongjian Shen ◽  
...  
Keyword(s):  
Kinetic Parameters ◽  
Three Dimensional ◽  
Large Family ◽  
Dimensional Structure ◽  
Glutathione S Transferase ◽  
X Ray Crystallography ◽  
Xenobiotic Exposure ◽  
Multifunctional Enzymes ◽  
Glutathione S Transferases ◽  
And Function

Arthropod Glutathione S-transferases (GSTs) constitute a large family of multifunctional enzymes that are mainly associated with xenobiotic or stress adaptation. GST-mediated xenobiotic adaptation is through direct metabolism or sequestration of xenobiotics, and/or indirectly by providing protection against oxidative stress induced by xenobiotic exposure. To date, the roles of GSTs in xenobiotic adaptation in the Colorado potato beetle (CPB), a notorious agriculture pest of plants within Solanaceae have not been well studied. Here, we functionally expressed and characterized an unclassified-class GST, LdGSTu1. The three-dimensional structure of the LdGSTu1 was solved with a resolution up to 1.8 Å by x-ray crystallography. Recombinant LdGSTu1 was used to determine enzyme activity and kinetic parameters using 1-chloro-2,4-dinitrobenzene (CDNB), GSH, p-nitrophenyl acetate (PNA) as substrates. The enzyme kinetic parameters and enzyme-substrate interaction studies demonstrated that LdGSTu1 could catalyze the conjugation of GSH to both CDNB and PNA, with a higher turnover number for CDNB than PNA. The LdGSTu1 enzyme inhibition assays demonstrated that the enzymatic conjugation of GSH to CDNB could be inhibited by multiple pesticides, suggesting a potential function of LdGSTu1 in xenobiotic adaptation.

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