A drunken search in crystallization space

Fazio, Vincent J.; Peat, Thomas S.; Newman, Janet

doi:10.1107/s2053230x1401841x

Cited by 40 publications

(42 citation statements)

References 26 publications

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“…On a technical note, the results here demonstrate that Shh-N can benefit from Zn 2+ and Ca 2+ supplementation after purification, especially if these ions are desired for inhibitor-binding studies, screening experiments or crystallization trials. Notably, the millimolar concentrations of multivalent ions present in typical commercial broad screens (Fazio et al, 2014) can supplant Ca 2+ or can co-occupy substoichiometrically bound ion sites, as observed here for Mg 2+ . These effects can lead to an interaction interface that differs from the physiologically relevant one.…”

Section: Discussionmentioning

confidence: 51%

Structure of Sonic Hedgehog protein in complex with zinc(II) and magnesium(II) reveals ion-coordination plasticity relevant to peptide drug design

Bonn-Breach

Jenkins

et al. 2019

Acta Crystallogr D Struct Biol

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The Hedgehog pathway is an essential cell-signaling paradigm implicated in cancer tumorigenesis and the developmental disorder holoprosencephaly, making it an attractive target for therapeutic design. The N-terminal domain of the Sonic Hedgehog protein (Shh-N) is the essential signaling molecule in the Hedgehog pathway. In this role Shh-N interacts with its cognate membrane receptor Patched, as well as the regulatory proteins HHIP and CDO, by utilizing interfaces harboring one or more divalent ions. Here, the crystal structure of human Shh-N is presented at 1.43 Å resolution, representing a landmark in the characterization of this protein. The structure reveals that the conserved Zn 2+binding site adopts an atypical octahedral coordination geometry, whereas an adjacent binding site, normally occupied by binuclear Ca 2+ , has been supplanted by a single octahedrally bound Mg 2+ . Both divalent sites are compared with those in previous Shh-N structures, which demonstrates a significant degree of plasticity of the Shh-N protein in terms of divalent ion binding. The presence of a high Mg 2+ concentration in the crystallization medium appears to have influenced metal loading at both metal ion-binding sites. These observations have technical and design implications for efforts focused on the development of inhibitors that target Shh-N-mediated protein-protein interactions.

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Section: Discussionmentioning

confidence: 51%

Structure of Sonic Hedgehog protein in complex with zinc(II) and magnesium(II) reveals ion-coordination plasticity relevant to peptide drug design

Bonn-Breach

Jenkins

et al. 2019

Acta Crystallogr D Struct Biol

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“…Initial experiments (150 nl protein solution plus 150 nl reservoir solution equilibrated against 50 ml reservoir solution) were set up with the protein at 5 mg ml À1 in 50 mM sodium chloride, 50 mM bis-Tris pH 6.5 with 1 mM lactulose with or without thrombin (100 ml protein solution added to 10 mg thrombin). Screens tested included Shotgun (Fazio et al, 2014) at both 20 and 8 C, as well as PACT and PSgradient both at 20 C. Detailed descriptions of these screens are available from the C6 website (http://c6.csiro.au; Newman et al, 2010). Spherulites from the (thrombin) trials were optimized using a combination of fine screening, microseeding and the substitution of either trypsin or chymotrypsin for the thrombin protease.…”

Section: Crystallizationmentioning

confidence: 99%

Structures of the transcriptional regulator BgaR, a lactose sensor

Newman

Caron

Nebl

et al. 2019

Acta Cryst Sect D Struct Biol

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The structure of BgaR, a transcriptional regulator of the lactose operon in Clostridium perfringens, has been solved by SAD phasing using a mercury derivative. BgaR is an exquisite sensor of lactose, with a binding affinity in the low-micromolar range. This sensor and regulator has been captured bound to lactose and to lactulose as well as in a nominal apo form, and was compared with AraC, another saccharide-binding transcriptional regulator. It is shown that the saccharides bind in the N-terminal region of a jelly-roll fold, but that part of the saccharide is exposed to bulk solvent. This differs from the classical AraC saccharide-binding site, which is mostly sequestered from the bulk solvent. The structures of BgaR bound to lactose and to lactulose highlight how specific and nonspecific interactions lead to a higher binding affinity of BgaR for lactose compared with lactulose. Moreover, solving multiple structures of BgaR in different space groups, both bound to saccharides and unbound, verified that the dimer interface along a C-terminal helix is similar to the dimer interface observed in AraC.

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“…Owing to its ability to promote protein-protein association (McPherson, 1976;Brzozowski & Tolley, 1994;George & Wilson, 1994;Vivarè s & Bonneté, 2002;Budayova et al, 1999;Tanaka & Ataka, 2002;Tanaka et al, 2003;Kulkarni et al, 2000), polyethylene glycol (PEG) has been widely used as one of the most effective precipitants in protein crystallization (Fazio et al, 2014). Various degrees of polymerization and additional modifications generate a large variety of PEG variants with molecular weights that range from 200 to >20 000 Da.…”

Section: Introductionmentioning

confidence: 99%

Defined PEG smears as an alternative approach to enhance the search for crystallization conditions and crystal-quality improvement in reduced screens

Chaikuad

Knapp

Delft

2015

Acta Crystallogr D Biol Crystallogr

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The quest for an optimal limited set of effective crystallization conditions remains a challenge in macromolecular crystallography, an issue that is complicated by the large number of chemicals which have been deemed to be suitable for promoting crystal growth. The lack of rational approaches towards the selection of successful chemical space and representative combinations has led to significant overlapping conditions, which are currently present in a multitude of commercially available crystallization screens. Here, an alternative approach to the sampling of widely used PEG precipitants is suggested through the use of PEG smears, which are mixtures of different PEGs with a requirement of either neutral or cooperatively positive effects of each component on crystal growth. Four newly defined smears were classified by molecular-weight groups and enabled the preservation of specific properties related to different polymer sizes. These smears not only allowed a wide coverage of properties of these polymers, but also reduced PEG variables, enabling greater sampling of other parameters such as buffers and additives. The efficiency of the smear-based screens was evaluated on more than 220 diverse recombinant human proteins, which overall revealed a good initial crystallization success rate of nearly 50%. In addition, in several cases successful crystallizations were only obtained using PEG smears, while various commercial screens failed to yield crystals. The defined smears therefore offer an alternative approach towards PEG sampling, which will benefit the design of crystallization screens sampling a wide chemical space of this key precipitant.

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A drunken search in crystallization space

Cited by 40 publications

References 26 publications

Structure of Sonic Hedgehog protein in complex with zinc(II) and magnesium(II) reveals ion-coordination plasticity relevant to peptide drug design

Structure of Sonic Hedgehog protein in complex with zinc(II) and magnesium(II) reveals ion-coordination plasticity relevant to peptide drug design

Structures of the transcriptional regulator BgaR, a lactose sensor

Defined PEG smears as an alternative approach to enhance the search for crystallization conditions and crystal-quality improvement in reduced screens

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