scholarly journals Low-resolution structure of the soluble domain GPAA1 (yGPAA170–247) of the glycosylphosphatidylinositol transamidase subunit GPAA1 from Saccharomyces cerevisiae

2013 ◽  
Vol 33 (2) ◽  
Author(s):  
Wuan Geok Saw ◽  
Birgit Eisenhaber ◽  
Frank Eisenhaber ◽  
Gerhard Grüber
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Solution Structure ◽  
Scattering Data ◽  
Radius Of Gyration ◽  
Low Resolution ◽  
X Ray Scattering ◽  
Eukaryotic Proteins ◽  
Structural Content ◽  
Α Helix ◽  
Gpi Transamidase

The GPI (glycosylphosphatidylinositol) transamidase complex catalyses the attachment of GPI anchors to eukaryotic proteins in the lumen of ER (endoplasmic reticulum). The Saccharomyces cerevisiae GPI transamidase complex consists of the subunits yPIG-K (Gpi8p), yPIG-S (Gpi17p), yPIG-T (Gpi16p), yPIG-U (CDC91/GAB1) and yGPAA1. We present the production of the two recombinant proteins yGPAA170–247 and yGPAA170–339 of the luminal domain of S. cerevisiae GPAA1, covering the amino acids 70–247 and 70–339 respectively. The secondary structural content of the stable and monodisperse yGPAA170–247 has been determined to be 28% α-helix and 27% β-sheet. SAXS (small-angle X-ray scattering) data showed that yGPAA170–247 has an Rg (radius of gyration) of 2.72±0.025 nm and Dmax (maximum dimension) of 9.14 nm. These data enabled the determination of the two domain low-resolution solution structure of yGPAA170–247. The large elliptical shape of yGPAA170–247 is connected via a short stalk to the smaller hook-like domain of 0.8 nm in length and 3.5 nm in width. The topological arrangement of yGPAA170–247 will be discussed together with the recently determined low-resolution structures of yPIG-K24–337 and yPIG-S38–467 from S. cerevisiae in the GPI transamidase complex.

scholarly journals The solution structure of the complement deregulator FHR5 reveals a compact dimer and provides new insights into CFHR5 nephropathy

2020 ◽  
Vol 295 (48) ◽  
pp. 16342-16358
Author(s):  
Nilufar Kadkhodayi-Kholghi ◽  
Jayesh S. Bhatt ◽  
Jayesh Gor ◽  
Lindsay C. McDermott ◽  
Daniel P. Gale ◽  
...  
Keyword(s):  
Solution Structure ◽  
Factor H ◽  
Scattering Data ◽  
Radius Of Gyration ◽  
Domain Pair ◽  
Complement Factor ◽  
X Ray ◽  
X Ray Scattering ◽  
Complement Regulator ◽  
Ray Scattering

The human complement Factor H–related 5 protein (FHR5) antagonizes the main circulating complement regulator Factor H, resulting in the deregulation of complement activation. FHR5 normally contains nine short complement regulator (SCR) domains, but a FHR5 mutant has been identified with a duplicated N-terminal SCR-1/2 domain pair that causes CFHR5 nephropathy. To understand how this duplication causes disease, we characterized the solution structure of native FHR5 by analytical ultracentrifugation and small-angle X-ray scattering. Sedimentation velocity and X-ray scattering indicated that FHR5 was dimeric, with a radius of gyration (Rg) of 5.5 ± 0.2 nm and a maximum protein length of 20 nm for its 18 domains. This result indicated that FHR5 was even more compact than the main regulator Factor H, which showed an overall length of 26–29 nm for its 20 SCR domains. Atomistic modeling for FHR5 generated a library of 250,000 physically realistic trial arrangements of SCR domains for scattering curve fits. Only compact domain structures in this library fit well to the scattering data, and these structures readily accommodated the extra SCR-1/2 domain pair present in CFHR5 nephropathy. This model indicated that mutant FHR5 can form oligomers that possess additional binding sites for C3b in FHR5. We conclude that the deregulation of complement regulation by the FHR5 mutant can be rationalized by the enhanced binding of FHR5 oligomers to C3b deposited on host cell surfaces. Our FHR5 structures thus explained key features of the mechanism and pathology of CFHR5 nephropathy.

scholarly journals Guinier peak analysis for visual and automated inspection of small-angle X-ray scattering data

2016 ◽  
Vol 49 (5) ◽  
pp. 1412-1419 ◽  
Author(s):  
Christopher D. Putnam
Keyword(s):  
Small Angle ◽  
Peak Position ◽  
Scattering Data ◽  
Radius Of Gyration ◽  
Automated Inspection ◽  
Elongation Ratio ◽  
Saxs Data ◽  
X Ray ◽  
X Ray Scattering ◽  
Ray Scattering

The Guinier region in small-angle X-ray scattering (SAXS) defines the radius of gyration,Rg, and the forward scattering intensity,I(0). In Guinier peak analysis (GPA), the plot ofqI(q)versus q2transforms the Guinier region into a characteristic peak for visual and automated inspection of data. Deviations of the peak position from the theoretical position in dimensionless GPA plots can suggest parameter errors, problematic low-resolution data, some kinds of intermolecular interactions or elongated scatters. To facilitate automated analysis by GPA, the elongation ratio (ER), which is the ratio of the areas in the pair-distribution functionP(r) after and before theP(r) maximum, was characterized; symmetric samples have ER values around 1, and samples with ER values greater than 5 tend to be outliers in GPA analysis. Use of GPA+ER can be a helpful addition to SAXS data analysis pipelines.

scholarly journals Solution structure and assembly of β-amylase2 from Arabidopsis thaliana

2019 ◽  
Author(s):  
Nithesh P. Chandrasekharan ◽  
Claire M. Ravenburg ◽  
Ian R. Roy ◽  
Jonathan D. Monroe ◽  
Christopher E. Berndsen
Keyword(s):  
Arabidopsis Thaliana ◽  
Ipomoea Batatas ◽  
Solution Structure ◽  
Scattering Data ◽  
Controlled Synthesis ◽  
X Ray ◽  
Domain Structures ◽  
X Ray Scattering ◽  
Loop Sequence ◽  
Assembly Analysis

AbstractStarch is a key energy storage molecule in plants that requires controlled synthesis and breakdown for effective plant growth. β-amylases (BAMs) hydrolyze starch into maltose to help meet the metabolic needs of the plant. In the model plant, Arabidopsis thaliana, there are nine BAMs which have apparently distinct functional and domain structures, although the functions of only a few of the BAMs are known and there are no 3-D structures of BAMs from this organism. Recently, AtBAM2 was proposed to form a tetramer based on chromatography and activity assays of mutants, however there was no direct observation of this tetramer. We collected small-angle X-ray scattering data on AtBAM2 and N-terminal mutants to describe the structure and assembly of the tetramer. Comparison of the scattering of the AtBAM2 tetramer to data collected using the sweet potato (Ipomoea batatas) BAM5, which is also reported to form a tetramer, showed there were differences in the overall assembly. Analysis of N-terminal truncations of AtBAM2 identified a loop sequence found only in BAM2 orthologs that appears to be critical for AtBAM2 tetramer assembly as well as activity.

Determination of small alterations in the radius of gyration by small-angle X-ray scattering

1971 ◽  
Vol 4 (4) ◽  
pp. 317-318 ◽  
Author(s):  
I. Simon
Keyword(s):  
Small Angle ◽  
Direct Determination ◽  
Differential Method ◽  
Scattering Data ◽  
Radius Of Gyration ◽  
X Ray ◽  
X Ray Scattering ◽  
Colloid Particles ◽  
Ray Scattering

A differential method is presented for the evaluation of small-angle X-ray scattering data. The direct determination of slight changes in the radius of gyration of colloid particles is rendered possible by the method proposed.

Low-resolution structure of immunoglobulins IgG1, IgM and rheumatoid factor IgM-RF from solution X-ray scattering data

2003 ◽  
Vol 36 (3) ◽  
pp. 503-508 ◽  
Author(s):  
Vladimir V. Volkov ◽  
Viktor A. Lapuk ◽  
Renata L. Kayushina ◽  
Eleonora V. Shtykova ◽  
Elena Yu. Varlamova ◽  
...  
Keyword(s):  
Rheumatoid Factor ◽  
Scattering Data ◽  
Low Resolution ◽  
X Ray ◽  
X Ray Scattering ◽  
Resolution Structure ◽  
Ray Scattering

Masking of the Fc region in human IgG4 by constrained X-ray scattering modelling: implications for antibody function and therapy

2010 ◽  
Vol 432 (1) ◽  
pp. 101-114 ◽  
Author(s):  
Yuki Abe ◽  
Jayesh Gor ◽  
Daniel G. Bracewell ◽  
Stephen J. Perkins ◽  
Paul A. Dalby
Keyword(s):  
Concentration Dependence ◽  
Solution Structure ◽  
Analytical Ultracentrifugation ◽  
Sedimentation Coefficient ◽  
Radius Of Gyration ◽  
X Ray ◽  
X Ray Scattering ◽  
Starting Point ◽  
Self Association ◽  
Ray Scattering

Of the four human IgG antibody subclasses IgG1–IgG4, IgG4 is of interest in that it does not activate complement and exhibits atypical self-association, including the formation of bispecific antibodies. The solution structures of antibodies are critical to understand function and therapeutic applications. Thus IgG4 was studied by synchrotron X-ray scattering. The Guinier X-ray radius of gyration RG increased from 5.0 nm to 5.1 nm with an increase of concentration. The distance distribution function P(r) revealed a single peak at 0.3 mg/ml, which resolved into two peaks that shifted to smaller r values at 1.3 mg/ml, even though the maximum dimension of IgG4 was unchanged at 17 nm. This indicated a small concentration dependence of the IgG4 solution structure. By analytical ultracentrifugation, no concentration dependence in the sedimentation coefficient of 6.4 S was observed. Constrained scattering modelling resulted in solution structural determinations that showed that IgG4 has an asymmetric solution structure in which one Fab–Fc pair is closer together than the other pair, and the accessibility of one side of the Fc region is masked by the Fab regions. The averaged distances between the two Fab–Fc pairs change by 1–2 nm with the change in IgG4 concentration. The averaged conformation of the Fab regions appear able to hinder complement C1q binding to the Fc region and the self-association of IgG4 through the Fc region. The present results clarify IgG4 function and provide a starting point to investigate antibody stability.

scholarly journals Solution structure and assembly of β-amylase 2 from Arabidopsis thaliana

2020 ◽  
Vol 76 (4) ◽  
pp. 357-365 ◽  
Author(s):  
Nithesh P. Chandrasekharan ◽  
Claire M. Ravenburg ◽  
Ian R. Roy ◽  
Jonathan D. Monroe ◽  
Christopher E. Berndsen
Keyword(s):  
Arabidopsis Thaliana ◽  
Ipomoea Batatas ◽  
Solution Structure ◽  
Scattering Data ◽  
Controlled Synthesis ◽  
X Ray ◽  
Domain Structures ◽  
X Ray Scattering ◽  
Loop Sequence ◽  
Assembly Analysis

Starch is a key energy-storage molecule in plants that requires controlled synthesis and breakdown for effective plant growth. β-Amylases (BAMs) hydrolyze starch into maltose to help to meet the metabolic needs of the plant. In the model plant Arabidopsis thaliana there are nine BAMs, which have apparently distinct functional and domain structures, although the functions of only a few of the BAMs are known and there are no 3D structures of BAMs from this organism. Recently, AtBAM2 was proposed to form a tetramer based on chromatography and activity assays of mutants; however, there was no direct observation of this tetramer. Here, small-angle X-ray scattering data were collected from AtBAM2 and its N-terminal truncations to describe the structure and assembly of the tetramer. Comparison of the scattering of the AtBAM2 tetramer with data collected from sweet potato (Ipomoea batatas) BAM5, which is also reported to form a tetramer, showed there were differences in the overall assembly. Analysis of the N-terminal truncations of AtBAM2 identified a loop sequence found only in BAM2 orthologs that appears to be critical for AtBAM2 tetramer assembly as well as for activity.

scholarly journals Small-Angle X-Ray Scattering Analysis of the Bifunctional Antibiotic Resistance Enzyme Aminoglycoside (6′) Acetyltransferase-Ie/Aminoglycoside (2″) Phosphotransferase-Ia Reveals a Rigid Solution Structure

2012 ◽  
Vol 56 (4) ◽  
pp. 1899-1906 ◽  
Author(s):  
Shane J. Caldwell ◽  
Albert M. Berghuis
Keyword(s):  
Small Angle ◽  
Active Sites ◽  
Solution Structure ◽  
Coenzyme A ◽  
Radius Of Gyration ◽  
Bifunctional Enzyme ◽  
X Ray ◽  
X Ray Scattering ◽  
The Impact ◽  
Ray Scattering

ABSTRACTAminoglycoside (6′) acetyltransferase-Ie/aminoglycoside (2″) phosphotransferase-Ia [AAC(6′)-Ie/APH(2″)-Ia] is one of the most problematic aminoglycoside resistance factors in clinical pathogens, conferring resistance to almost every aminoglycoside antibiotic available to modern medicine. Despite 3 decades of research, our understanding of the structure of this bifunctional enzyme remains limited. We used small-angle X-ray scattering (SAXS) to model the structure of this bifunctional enzyme in solution and to study the impact of substrate binding on the enzyme. It was observed that the enzyme adopts a rigid conformation in solution, where the N-terminal AAC domain is fixed to the C-terminal APH domain and not loosely tethered. The addition of acetyl-coenzyme A, coenzyme A, GDP, guanosine 5′-[β,γ-imido]triphosphate (GMPPNP), and combinations thereof to the protein resulted in only modest changes to the radius of gyration (RG) of the enzyme, which were not consistent with any large changes in enzyme structure upon binding. These results imply some selective advantage to the bifunctional enzyme beyond coexpression as a single polypeptide, likely linked to an improvement in enzymatic properties. We propose that the rigid structure contributes to improved electrostatic steering of aminoglycoside substrates toward the two active sites, which may provide such an advantage.

scholarly journals Structure of the bifunctional aminoglycoside-resistance enzyme AAC(6′)-Ie-APH(2′′)-Ia revealed by crystallographic and small-angle X-ray scattering analysis

2014 ◽  
Vol 70 (10) ◽  
pp. 2754-2764 ◽  
Author(s):  
Clyde A. Smith ◽  
Marta Toth ◽  
Thomas M. Weiss ◽  
Hilary Frase ◽  
Sergei B. Vakulenko
Keyword(s):  
Small Angle ◽  
Coenzyme A ◽  
Full Length ◽  
Scattering Data ◽  
Bifunctional Enzyme ◽  
Aminoglycoside Resistance ◽  
X Ray ◽  
X Ray Scattering ◽  
Α Helix ◽  
Ray Scattering

Broad-spectrum resistance to aminoglycoside antibiotics in clinically important Gram-positive staphylococcal and enterococcal pathogens is primarily conferred by the bifunctional enzyme AAC(6′)-Ie-APH(2′′)-Ia. This enzyme possesses an N-terminal coenzyme A-dependent acetyltransferase domain [AAC(6′)-Ie] and a C-terminal GTP-dependent phosphotransferase domain [APH(2′′)-Ia], and together they produce resistance to almost all known aminoglycosides in clinical use. Despite considerable effort over the last two or more decades, structural details of AAC(6′)-Ie-APH(2′′)-Ia have remained elusive. In a recent breakthrough, the structure of the isolated C-terminal APH(2′′)-Ia enzyme was determined as the binary Mg2GDP complex. Here, the high-resolution structure of the N-terminal AAC(6′)-Ie enzyme is reported as a ternary kanamycin/coenzyme A abortive complex. The structure of the full-length bifunctional enzyme has subsequently been elucidated based upon small-angle X-ray scattering data using the two crystallographic models. The AAC(6′)-Ie enzyme is joined to APH(2′′)-Ia by a short, predominantly rigid linker at the N-terminal end of a long α-helix. This α-helix is in turn intrinsically associated with the N-terminus of APH(2′′)-Ia. This structural arrangement supports earlier observations that the presence of the intact α-helix is essential to the activity of both functionalities of the full-length AAC(6′)-Ie-APH(2′′)-Ia enzyme.

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