Crystallization of protein–ligand complexes

Hassell, Anne M.; An, Gang; Bledsoe, Randy K.; Bynum, Jane; Carter, H. Luke; Deng, Su-Jun; Gampe, Robert T.; Grisard, Tamara E.; Madauss, Kevin P.; Nolte, R.T.; Rocque, Warren J.; Wang, Liping; Weaver, K.; Williams, Shawn P.; Wisely, G. Bruce; Xu, Renfeng; Shewchuk, Lisa M.

doi:10.1107/s0907444906047020

Cited by 131 publications

(93 citation statements)

References 12 publications

(14 reference statements)

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“…For data collection, native crystals were transferred to cryoprotectant solutions consisting of mother liquor plus 20% (v/v) glycerol before being cooled to 100 K in liquid nitrogen. Complexes with the natural substrates melibiose (Sigma) and raffinose (Sigma) were obtained by the soaking method (11). To minimize crystal manipulation during the soaking with the substrates, drop solution was substituted with a stabilizing solution (20% PEG 3350, 0.1 M Bistris propane, pH 8.5, 0.2 M KSCN) containing the substrate at 50 mM concentration, incubated for 5 min, and then substituted again with cryoprotectant solution in which the PEG 3350 concentration was increased to 35%.…”

Section: Methodsmentioning

confidence: 99%

Structural Analysis of Saccharomyces cerevisiae α-Galactosidase and Its Complexes with Natural Substrates Reveals New Insights into Substrate Specificity of GH27 Glycosidases

Fernández-Leiro

Pereira-Rodríguez

Cerdán

et al. 2010

Journal of Biological Chemistry

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␣-Galactosidases catalyze the hydrolysis of terminal ␣-1,6-galactosyl units from galacto-oligosaccharides and polymeric galactomannans. The crystal structures of tetrameric Saccharomyces cerevisiae ␣-galactosidase and its complexes with the substrates melibiose and raffinose have been determined to 1.95, 2.40, and 2.70 Å resolution. The monomer folds into a catalytic (␣/␤) 8 barrel and a C-terminal ␤-sandwich domain with unassigned function. This pattern is conserved with other family 27 glycosidases, but this enzyme presents a unique 45-residue insertion in the ␤-sandwich domain that folds over the barrel protecting it from the solvent and likely explaining its high stability. The structure of the complexes and the mutational analysis show that oligomerization is a key factor in substrate binding, as the substrates are located in a deep cavity making direct interactions with the adjacent subunit. Furthermore, docking analysis suggests that the supplementary domain could be involved in binding sugar units distal from the scissile bond, therefore ascribing a role in fine-tuning substrate specificity to this domain. It may also have a role in promoting association with the polymeric substrate because of the ordered arrangement that the four domains present in one face of the tetramer. Our analysis extends to other family 27 glycosidases, where some traits regarding specificity and oligomerization can be formulated on the basis of their sequence and the structures available. These results improve our knowledge on the activity of this important family of enzymes and give a deeper insight into the structural features that rule modularity and protein-carbohydrate interactions.Galactose is present in the oligosaccharides of many plant seeds and is also essential in structures as the hemicelluloses. These polymers build up the plant cell wall and represent a huge storage of carbon within the biosphere and might be an important source of renewable energy (1). In humans, mutations of the ␣-galactosidase gene cause incomplete degradation of glycolipids and glycoproteins, resulting in Fabry disease (2). Different strategies involving recombinant ␣-galactosidases are being developed for the treatment of this disease. Another interesting application is the conversion between the ABO blood groups, determined by differences in polysaccharide structures present in the surface of red blood cells. Some ␣-galactosidases are able to remove the ␣-linked terminal galactose that differs between O antigen (universal blood) and B antigen, and processes involving plant ␣-galactosidases are being developed to obtain O-type blood from B-type donors (3). Furthermore, the activity of ␣-galactosidase is of great interest in many biotechnological applications. It is used to improve the quality and yield of sucrose in the sugar beet industry by achieving an efficient raffinose and other galacto-oligosaccharide hydrolyses. In addition, the processing of soybean-related products and other legume-derived food with this enzyme reduces the content of...

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Section: Methodsmentioning

confidence: 99%

Structural Analysis of Saccharomyces cerevisiae α-Galactosidase and Its Complexes with Natural Substrates Reveals New Insights into Substrate Specificity of GH27 Glycosidases

Fernández-Leiro

Pereira-Rodríguez

Cerdán

et al. 2010

Journal of Biological Chemistry

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“…A 5 ′ -UUAGG-3 ′ RNA ligand was introduced into the crystals by the soaking method (Hassell et al 2007;Reiter et al 2010). Other oligonucleotides were tested in crystal soaking trials and include: the 53-and 25-nt GQ RNA, as well as UUAGG, UUUUU, CCCUAA, UUAGGAG, AAAAAGCAAA, and GGUUUUUCUUUU RNAs.…”

Section: Crystallization Of Lsd1-corest Complexmentioning

confidence: 99%

G-quadruplex RNA binding and recognition by the lysine-specific histone demethylase-1 enzyme

Hirschi

Martin

Luka

et al. 2016

RNA

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Lysine-specific histone demethylase 1 (LSD1) is an essential epigenetic regulator in metazoans and requires the co-repressor element-1 silencing transcription factor (CoREST) to efficiently catalyze the removal of mono-and dimethyl functional groups from histone 3 at lysine positions 4 and 9 (H3K4/9). LSD1 interacts with over 60 regulatory proteins and also associates with lncRNAs (TERRA, HOTAIR), suggesting a regulatory role for RNA in LSD1 function. We report that a stacked, intramolecular G-quadruplex (GQ) forming TERRA RNA (GG[UUAGGG] 8 UUA) binds tightly to the functional LSD1-CoREST complex (K d ≈ 96 nM), in contrast to a single GQ RNA unit ([UUAGGG] 4 U), a GQ DNA ([TTAGGG] 4 T), or an unstructured single-stranded RNA. Stabilization of a parallel-stranded GQ RNA structure by monovalent potassium ions (K + ) is required for high affinity binding to the LSD1-CoREST complex. These data indicate that LSD1 can distinguish between RNA and DNA as well as structured versus unstructured nucleotide motifs. Further, cross-linking mass spectrometry identified the primary location of GQ RNA binding within the SWIRM/amine oxidase domain (AOD) of LSD1. An ssRNA binding region adjacent to this GQ binding site was also identified via X-ray crystallography. This RNA binding interface is consistent with kinetic assays, demonstrating that a GQ-forming RNA can serve as a noncompetitive inhibitor of LSD1-catalyzed demethylation. The identification of a GQ RNA binding site coupled with kinetic data suggests that structured RNAs can function as regulatory molecules in LSD1-mediated mechanisms.

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“…One way is to crystallize a large component and then soak the protein with the other components or molecules (Hassell et al, 2007). This strategy is usually applied to the structures of proteins in complex with small molecules.…”

Section: Discussionmentioning

confidence: 99%

An efficient method to produce recombinant bacterial effector Tir coupled cytoskeleton protein (TCCP) complexed with host protein insulin receptor tyrosine kinase substrate (IRTKS)

Yao¹,

Jiang²,

Wang³

et al. 2013

Afr. J. Microbiol. Res.

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An important pathogenic process in enterohemorrhagic Escherichia coli (EHEC) associated diseases is the formation of attaching and effacing ( (SH3 IRTKS ) to the proline rich repeat (PRR) domain of TCCP , which is important for the induction of A/E lesions. The inability to efficiently produce the purified target complex has hindered the structural determination of these protein complexes. Here, we report an effective method for the generation of a complex consisting of SH3 IRTKS with three PRR TCCP domains. Two recombinant fragments, TCCP3R and TCCP5R, as well as SH3 IRTKS , were successfully expressed in soluble form and purified. In addition, methods were established to prepare two different protein complexes, SH3 IRTKS -TCCP3R and SH3 IRTKS -TCCP5R. These methods are a good foundation for future studies on the crystal structure of TCCP-IRTKS. Meanwhile, recombinant TCCP was shown to directly bind to IRTKS in vitro, which provides additional evidence for the interaction between these two molecules.

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Crystallization of protein–ligand complexes

Cited by 131 publications

References 12 publications

Structural Analysis of Saccharomyces cerevisiae α-Galactosidase and Its Complexes with Natural Substrates Reveals New Insights into Substrate Specificity of GH27 Glycosidases

Structural Analysis of Saccharomyces cerevisiae α-Galactosidase and Its Complexes with Natural Substrates Reveals New Insights into Substrate Specificity of GH27 Glycosidases

G-quadruplex RNA binding and recognition by the lysine-specific histone demethylase-1 enzyme

An efficient method to produce recombinant bacterial effector Tir coupled cytoskeleton protein (TCCP) complexed with host protein insulin receptor tyrosine kinase substrate (IRTKS)

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