Trapping Transient Protein–Protein Interactions in Polyketide Biosynthesis

Schnarr, Nathan A.; Khosla, Chaitan

doi:10.1021/cb600451d

Cited by 5 publications

(3 citation statements)

References 16 publications

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“…Weak protein–protein complexes, which may readily dissociate or associate depending on precise physiological condition or environment, play an important role in many biological processes, such as signal transduction71 electron transport,72–74 transcriptional regulation,75, 76 growth factors,77–80 molecular switches,81–83 cell–cell recognition,84 and many others 85–99. The dissociation constant K D of weak complexes may reach a few hundred μM,90 which corresponds to Δ G 0 of only a few kcal/mol.…”

Section: Resultsmentioning

confidence: 99%

Crystal contacts as nature's docking solutions

2009

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The assumption that crystal contacts reflect natural macromolecular interactions makes a basis for many studies in structural biology. However, the crystal state may correspond to a global minimum of free energy where biologically relevant interactions are sacrificed in favor to unspecific contacts. A large-scale docking experiment was performed to assess the extent of misrepresentation of natural (in-solvent) protein dimers by crystal packing. As found, the failure rate of docking may be quantitatively interpreted if both calculation errors and misrepresentation effects are taken into account. The failure rate analysis is based on the assumption that crystal structures reflect thermodynamic equilibrium between different dimeric configurations. The analysis gives an estimate of misrepresentation probability, which suggests that weakly bound complexes with K(D) > or = 100 microM (some 20% of all dimers in the PDB) have higher than 50% chances to be misrepresented by crystals. The developed theoretical framework is applicable in other studies, where experimental results may be viewed as snapshots of systems in thermodynamic equilibrium.

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Section: Resultsmentioning

confidence: 99%

Crystal contacts as nature's docking solutions

2009

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“…it may vary depending on the external conditions. Protein-protein complexes, which may dissociate or associate depending on the biochemical environment, play an important role in many biological processes, such as signal transduction (Gomperts et al, 2002), electron transport (Brown et al, 1999;Doyle et al, 1986;Ren et al, 1993), transcriptional regulation (Huang et al, 1997;Sengchanthalangsy et al, 1999), growth factors (Lu et al, 1995;Hsu et al, 1997;Bianchet et al, 2000;Blundell et al, 2000), molecular switches (Darling et al, 2000;Pan & Heitman, 2002;Ma & Karplus, 1997), cell-cell recognition (Alattia et al, 1997) and many others (Waas & Dalby, 2002;Cho et al, 2006;Johannes, 2007;Bonet et al, 2006;Ansari & Helms, 2005;Nooren & Thornton, 2003;Schnarr & Khosla, 2006;Vaynberg & Qin, 2006;Fuentes et al, 2006Fuentes et al, , 2007Boelens et al, 1991;Ceres & Zlotnick, 2002;Buts et al, 2001;Nyfeler et al, 2005;Hamelryck et al, 2000). The dissociation constant K d of weak complexes may reach a few hundred mM (Nooren & Thornton, 2003), which corresponds to a ÁG diss of only a few kcal mol…”

Section: General Backgroundmentioning

confidence: 99%

Macromolecular complexes in crystals and solutions

Krissinel

2011

Acta Crystallogr D Biol Crystallogr

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This paper presents a discussion of existing methods for the analysis of macromolecular interactions and complexes in crystal packing. Typical situations and conditions where wrong answers may be obtained in the course of ordinary procedures are presented and discussed. The more general question of what the relationship is between natural (in-solvent) and crystallized assemblies is discussed and researched. A computational analysis suggests that weak interactions with K d ! 100 mM have a considerable chance of being lost during the course of crystallization. In such instances, crystal packing misrepresents macromolecular complexes and interactions. For as many as 20% of protein dimers in the PDB the likelihood of misrepresentation is estimated to be higher than 50%. Given that weak macromolecular interactions play an important role in many biochemical processes, these results suggest that a complementary noncrystallographic study should be always conducted when inferring structural aspects of weakly bound complexes.

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“…Current approaches that have been used to effectively trap intermediates for structural analysis typically utilize an activity-based approach. While many successful examples exist, the disadvantages of such approaches are, firstly, that the intermediate isolated is not the natural one but rather a mimic that often lacks or distorts the enzyme active site and, secondly, that the synthesis of the crosslinking reagents is typically non-trivial [10][11][12][13][14][15][16][17][18][19][20][21][22]. Given this, we sought to develop a linking technique that could generate controlled protein-protein fusions that are linked via variable peptidebased linkers.…”

Section: Introductionmentioning

confidence: 99%

SNaPe: a versatile method to generate multiplexed protein fusions using synthetic linker peptides forin vitroapplications

Ulrich

Cryle

2016

J. Pept. Sci.

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Understanding the structure and function of protein complexes and multi-domain proteins is highly important in biology, although the in vitro characterization of these systems is often complicated by their size or the transient nature of protein/protein interactions. To assist in the characterization of such protein complexes, we have developed a modular approach to fusion protein generation that relies upon Sortase-mediated and Native chemical ligation using synthetic Peptide linkers (SNaPe) to link two separately expressed proteins. In this approach, we utilize two separate linking steps - sortase-mediated and native chemical ligation - together with a library of peptide linkers to generate libraries of fusion proteins. We have demonstrated the viability of SNaPe to generate libraries from fusion protein constructs taken from the biosynthetic enzymes responsible for late stage aglycone assembly during glycopeptide antibiotic biosynthesis. Crucially, SNaPe was able to generate fusion proteins that are inaccessible via direct expression of the fusion construct itself. This highlights the advantages of SNaPe to not only access fusion proteins that have been previously unavailable for biochemical and structural characterization but also to do so in a manner that enables the linker itself to be controlled as an experimental parameter of fusion protein generation. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

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Trapping Transient Protein–Protein Interactions in Polyketide Biosynthesis

Abstract: Transient biomolecular interactions are essential for biological processes, but strategies for studying them have remained elusive. A paper in this issue shows how natural enzymatic activities can be exploited to examine proteinprotein interactions in fatty acid synthase.

Cited by 5 publications

References 16 publications

Crystal contacts as nature's docking solutions

Crystal contacts as nature's docking solutions

Macromolecular complexes in crystals and solutions

SNaPe: a versatile method to generate multiplexed protein fusions using synthetic linker peptides forin vitroapplications

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