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Capturing the Structure of the Substrate Bound Condensation Domain
Please cite this article as: Kittila¨ et al., Capturing the Structure of the Substrate Bound Condensation Domain, Cell Chemical Biology (2016), http:// dx.doi.org/10.1016/j.chembiol.2016.03.003
Cell Chemical Biology
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Capturing the Structure of the Substrate Bound Condensation Domain Tiia Kittila¨1 and Max J. Cryle1,2,3,* 1Department
of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Jahnstrasse 29, 69120 Heidelberg, Germany Australia Monash University, Clayton, VIC 3800, Australia 3The Department of Biochemistry and Molecular Biology and ARC Centre of Excellence in Advanced Molecular Imaging, Monash University, 15 Innovation Walk, Clayton, VIC 3800, Australia *Correspondence: [email protected] http://dx.doi.org/10.1016/j.chembiol.2016.03.003 2EMBL
Condensation domains of nonribosomal peptide synthetase machineries have so far escaped detailed structural analysis. In this issue of Cell Chemical Biology, Bloudoff et al. (2016) describe a protein tethering technique that allowed the authors to obtain structural information on the substrate bound state of the first condensation domain from calcium-dependent antibiotic biosynthesis, thus opening a new window into how these important biosynthetic machineries function. The importance of natural products in modern medicine is clear: of the many different classes of secondary metabolites, some of the most intriguing chemical structures—with equally important biological activities—are products of the mega-enzyme polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs). While both PKSs and NRPSs share similarities in their use of thiol-templated substrate tethering via carrier proteins, their domain architecture is quite distinct. NRPS systems are based around a module comprising three domains: adenylation (A) domains, which are responsible for amino acid selection and activation and are not limited to proteogenic amino acids; carrier protein (CP) domains, which serve as the attachment points for synthetic intermediates; and condensation (C) domains, where peptide bound formation between adjacent CP-bound substrates occurs (Figure 1A) (Hur et al., 2012). Many other catalytic domains, including those fused in cis as well as external domains acting in trans, supplement C-A-CP modules and allow NRPS systems to produce the vast array of chemical structures reported (Hur et al., 2012). With the complexity of NRPS assembly lines, the ability to structurally interrogate discrete states of the machinery is absolutely crucial to understanding NRPS function; this includes how domains move during catalysis, the roles of critical active site residues and the selection and enforcement of substrate selectivity during peptide synthesis. With some notable
exceptions, in particular that of the terminal module of Surfactin synthase from Marahiel and co-workers (Tanovic et al., 2008), the majority of highly informative multi-domain structures have been resolved with the aid of chemical tools to generate specific domain states, including the recent structures of NRPS domains in multiple catalytic states from the laboratories of Gulick (Drake et al., 2016) and Schmeing (Reimer et al., 2016), the structure of a thioesterase-CP structure from the Bruner laboratory (Liu et al., 2011), and a Cytochrome P450CP complex from skyllamycin biosynthesis (Haslinger et al., 2014). In all cases, these structures utilized chemical modifications that were tailored to trap specific states based upon knowledge of the enzymatic mechanism. This was combined with the ability to generate loaded forms of the CP domain by enzyme-catalyzed modification using synthetic substrates; particularly impressive in their impact have been the vinyl sulfonamide derivatives used to trap the CP-bound thiolation state of various adenylation domains (pioneered by the Aldrich group [Qiao et al., 2007]), whose use was highlighted in several structures from the Gulick laboratory (Drake et al., 2016). There is no doubt that the recent dramatic increase in highly informative, multi-domain structures from NRPS systems will prove a boon to researchers interested in both understanding and redesigning NRPS systems. However, there is one crucial catalytic domain
whose structural characterization remains limited—the condensation domain. This is not only due to the limited number of structures solved for C-domains, but also to the lack of substrate bound structures. Thus, the novel approach from the Schmeing laboratory that utilizes a substrate tethering approach to gain structural insights into the mechanism and substrate binding of C-domains fills the gaps in our understanding of NRPS biosynthesis; in particular, how C-domains control the stereochemistry of peptide synthesis and to what extent they play a role in proofreading A-domain selection (Bloudoff et al., 2016). In their approach, Bloudoff et al. utilized a model generated from their previous structural characterization of the first C-domain from calcium-dependent antibiotic (CDA) biosynthesis (Figure 1B). This allowed them to selectively introduce a cysteine mutation within the acceptor site of the C-domain in such a position as to covalently bind a small molecule mimic of the substrate loaded phosphopantetheinyl (PPE) arm of the acceptor carrier protein (Bloudoff et al., 2016). By using a modifying group with relatively low reactivity, they were able to use the apparent affinity of this PPE mimic for the C-domain to allow the specific labeling of the introduced cysteine residue. Modified C-domains were able to act as the acceptor for the upstream donor substrate and, more importantly, allowed the structure of such a modified C-domain to be solved. This revealed that a crucial active site histidine residue, whose exact role was
Cell Chemical Biology 23, March 17, 2016 ª2016 Elsevier Ltd All rights reserved 315
Please cite this article as: Kittila¨ et al., Capturing the Structure of the Substrate Bound Condensation Domain, Cell Chemical Biology (2016), http:// dx.doi.org/10.1016/j.chembiol.2016.03.003
Cell Chemical Biology
Previews
glycopeptide antibiotic oxidative crosslinking cascade, relies upon a catalytically inactive C-domain (Haslinger et al., 2015). Given the importance of C-domains and that these domains appear to play roles far exceeding previous expectations, the structural characterization of further trapped C-domain states in the future is a priority—many of these can now be envisaged using the approach pioneered by the Schmeing group (Bloudoff et al., 2016). It is also highly satisfying to see the importance of clever chemistry in understanding NRPS biosynthesis, a source of some of the most impressive chemical structures reported to date.
REFERENCES
Figure 1. Catalysis by a Condensation Domain from Nonribosomal Peptide Synthesis
Bloudoff, K., Alonzo, D.A., and Schmeing, T.M. (2016). Cell Chem. Biol. 23, this issue, 331–339.
(A) A typical reaction catalyzed by a condensation domain from nonribosomal peptide synthesis, in which the acceptor amino acid attacks the tethering thioester of the upstream donor substrate, resulting in transfer onto the acceptor via peptide bond formation. (B) A strategy adopted by Bloudoff et al. (2016), in which a mutated condensation domain (E17C) from CDA biosynthesis is covalently modified by donor substrate mimics (orange), which in turn allowed the first structure of a trapped condensation domain/donor substrate complex to be determined.
Drake, E.J., Miller, B.R., Shi, C., Tarrasch, J.T., Sundlov, J.A., Allen, C.L., Skiniotis, G., Aldrich, C.C., and Gulick, A.M. (2016). Nature 529, 235–238.
previously unclear, was hydrogen bonding to the amine of the tethered PPE mimic and thus is likely to be involved in substrate orientation within the C-domain active site rather than acting as a strong base or stabilizing the transition state (Bloudoff et al., 2016). Furthermore, in these structures it also was possible to directly observe which residues were in close proximity to the tethered PPE mimic and allow the role of several such residues to be assessed via site directed mutagenesis; these types of studies are crucial to reveal the source and degree of substrate selectivity of C-domains, which have previously evaded characterization (Bloudoff et al., 2016).
Haslinger, K., Brieke, C., Uhlmann, S., Sieverling, L., Su¨ssmuth, R.D., and Cryle, M.J. (2014). Angew. Chem. Int. Ed. Engl. 53, 8518–8522.
The ability to gain the first insights into substrate orientation in condensation domains is a vital step forward in our efforts to understand and redesign NRPS machineries. From recent studies, it has become clear that C-domains are not merely restricted to peptide bond formation. Townsend and coworkers have shown that the norcardicin b-lactam moiety is in fact installed by a C-domain (Gaudelli et al., 2015), one of the many stunning revelations from this laboratory concerning norcardicin biosynthesis. Our work on glycopeptide antibiotics has also demonstrated that the basis for P450 recruitment to the NRPS-bound peptide, which underpins the unique
316 Cell Chemical Biology 23, March 17, 2016 ª2016 Elsevier Ltd All rights reserved
Gaudelli, N.M., Long, D.H., and Townsend, C.A. (2015). Nature 520, 383–387.
Haslinger, K., Peschke, M., Brieke, C., Maximowitsch, E., and Cryle, M.J. (2015). Nature 521, 105–109. Hur, G.H., Vickery, C.R., and Burkart, M.D. (2012). Nat. Prod. Rep. 29, 1074–1098. Liu, Y., Zheng, T., and Bruner, S.D. (2011). Chem. Biol. 18, 1482–1488. Qiao, C., Wilson, D.J., Bennett, E.M., and Aldrich, C.C. (2007). J. Am. Chem. Soc. 129, 6350–6351. Reimer, J.M., Aloise, M.N., Harrison, P.M., and Schmeing, T.M. (2016). Nature 529, 239–242. Tanovic, A., Samel, S.A., Essen, L.-O., and Marahiel, M.A. (2008). Science 321, 659–663.
Cell Chemical Biology
Previews
Capturing the Structure of the Substrate Bound Condensation Domain Tiia Kittila¨1 and Max J. Cryle1,2,3,* 1Department
of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Jahnstrasse 29, 69120 Heidelberg, Germany Australia Monash University, Clayton, VIC 3800, Australia 3The Department of Biochemistry and Molecular Biology and ARC Centre of Excellence in Advanced Molecular Imaging, Monash University, 15 Innovation Walk, Clayton, VIC 3800, Australia *Correspondence: [email protected] http://dx.doi.org/10.1016/j.chembiol.2016.03.003 2EMBL
Condensation domains of nonribosomal peptide synthetase machineries have so far escaped detailed structural analysis. In this issue of Cell Chemical Biology, Bloudoff et al. (2016) describe a protein tethering technique that allowed the authors to obtain structural information on the substrate bound state of the first condensation domain from calcium-dependent antibiotic biosynthesis, thus opening a new window into how these important biosynthetic machineries function. The importance of natural products in modern medicine is clear: of the many different classes of secondary metabolites, some of the most intriguing chemical structures—with equally important biological activities—are products of the mega-enzyme polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs). While both PKSs and NRPSs share similarities in their use of thiol-templated substrate tethering via carrier proteins, their domain architecture is quite distinct. NRPS systems are based around a module comprising three domains: adenylation (A) domains, which are responsible for amino acid selection and activation and are not limited to proteogenic amino acids; carrier protein (CP) domains, which serve as the attachment points for synthetic intermediates; and condensation (C) domains, where peptide bound formation between adjacent CP-bound substrates occurs (Figure 1A) (Hur et al., 2012). Many other catalytic domains, including those fused in cis as well as external domains acting in trans, supplement C-A-CP modules and allow NRPS systems to produce the vast array of chemical structures reported (Hur et al., 2012). With the complexity of NRPS assembly lines, the ability to structurally interrogate discrete states of the machinery is absolutely crucial to understanding NRPS function; this includes how domains move during catalysis, the roles of critical active site residues and the selection and enforcement of substrate selectivity during peptide synthesis. With some notable
exceptions, in particular that of the terminal module of Surfactin synthase from Marahiel and co-workers (Tanovic et al., 2008), the majority of highly informative multi-domain structures have been resolved with the aid of chemical tools to generate specific domain states, including the recent structures of NRPS domains in multiple catalytic states from the laboratories of Gulick (Drake et al., 2016) and Schmeing (Reimer et al., 2016), the structure of a thioesterase-CP structure from the Bruner laboratory (Liu et al., 2011), and a Cytochrome P450CP complex from skyllamycin biosynthesis (Haslinger et al., 2014). In all cases, these structures utilized chemical modifications that were tailored to trap specific states based upon knowledge of the enzymatic mechanism. This was combined with the ability to generate loaded forms of the CP domain by enzyme-catalyzed modification using synthetic substrates; particularly impressive in their impact have been the vinyl sulfonamide derivatives used to trap the CP-bound thiolation state of various adenylation domains (pioneered by the Aldrich group [Qiao et al., 2007]), whose use was highlighted in several structures from the Gulick laboratory (Drake et al., 2016). There is no doubt that the recent dramatic increase in highly informative, multi-domain structures from NRPS systems will prove a boon to researchers interested in both understanding and redesigning NRPS systems. However, there is one crucial catalytic domain
whose structural characterization remains limited—the condensation domain. This is not only due to the limited number of structures solved for C-domains, but also to the lack of substrate bound structures. Thus, the novel approach from the Schmeing laboratory that utilizes a substrate tethering approach to gain structural insights into the mechanism and substrate binding of C-domains fills the gaps in our understanding of NRPS biosynthesis; in particular, how C-domains control the stereochemistry of peptide synthesis and to what extent they play a role in proofreading A-domain selection (Bloudoff et al., 2016). In their approach, Bloudoff et al. utilized a model generated from their previous structural characterization of the first C-domain from calcium-dependent antibiotic (CDA) biosynthesis (Figure 1B). This allowed them to selectively introduce a cysteine mutation within the acceptor site of the C-domain in such a position as to covalently bind a small molecule mimic of the substrate loaded phosphopantetheinyl (PPE) arm of the acceptor carrier protein (Bloudoff et al., 2016). By using a modifying group with relatively low reactivity, they were able to use the apparent affinity of this PPE mimic for the C-domain to allow the specific labeling of the introduced cysteine residue. Modified C-domains were able to act as the acceptor for the upstream donor substrate and, more importantly, allowed the structure of such a modified C-domain to be solved. This revealed that a crucial active site histidine residue, whose exact role was
Cell Chemical Biology 23, March 17, 2016 ª2016 Elsevier Ltd All rights reserved 315
Please cite this article as: Kittila¨ et al., Capturing the Structure of the Substrate Bound Condensation Domain, Cell Chemical Biology (2016), http:// dx.doi.org/10.1016/j.chembiol.2016.03.003
Cell Chemical Biology
Previews
glycopeptide antibiotic oxidative crosslinking cascade, relies upon a catalytically inactive C-domain (Haslinger et al., 2015). Given the importance of C-domains and that these domains appear to play roles far exceeding previous expectations, the structural characterization of further trapped C-domain states in the future is a priority—many of these can now be envisaged using the approach pioneered by the Schmeing group (Bloudoff et al., 2016). It is also highly satisfying to see the importance of clever chemistry in understanding NRPS biosynthesis, a source of some of the most impressive chemical structures reported to date.
REFERENCES
Figure 1. Catalysis by a Condensation Domain from Nonribosomal Peptide Synthesis
Bloudoff, K., Alonzo, D.A., and Schmeing, T.M. (2016). Cell Chem. Biol. 23, this issue, 331–339.
(A) A typical reaction catalyzed by a condensation domain from nonribosomal peptide synthesis, in which the acceptor amino acid attacks the tethering thioester of the upstream donor substrate, resulting in transfer onto the acceptor via peptide bond formation. (B) A strategy adopted by Bloudoff et al. (2016), in which a mutated condensation domain (E17C) from CDA biosynthesis is covalently modified by donor substrate mimics (orange), which in turn allowed the first structure of a trapped condensation domain/donor substrate complex to be determined.
Drake, E.J., Miller, B.R., Shi, C., Tarrasch, J.T., Sundlov, J.A., Allen, C.L., Skiniotis, G., Aldrich, C.C., and Gulick, A.M. (2016). Nature 529, 235–238.
previously unclear, was hydrogen bonding to the amine of the tethered PPE mimic and thus is likely to be involved in substrate orientation within the C-domain active site rather than acting as a strong base or stabilizing the transition state (Bloudoff et al., 2016). Furthermore, in these structures it also was possible to directly observe which residues were in close proximity to the tethered PPE mimic and allow the role of several such residues to be assessed via site directed mutagenesis; these types of studies are crucial to reveal the source and degree of substrate selectivity of C-domains, which have previously evaded characterization (Bloudoff et al., 2016).
Haslinger, K., Brieke, C., Uhlmann, S., Sieverling, L., Su¨ssmuth, R.D., and Cryle, M.J. (2014). Angew. Chem. Int. Ed. Engl. 53, 8518–8522.
The ability to gain the first insights into substrate orientation in condensation domains is a vital step forward in our efforts to understand and redesign NRPS machineries. From recent studies, it has become clear that C-domains are not merely restricted to peptide bond formation. Townsend and coworkers have shown that the norcardicin b-lactam moiety is in fact installed by a C-domain (Gaudelli et al., 2015), one of the many stunning revelations from this laboratory concerning norcardicin biosynthesis. Our work on glycopeptide antibiotics has also demonstrated that the basis for P450 recruitment to the NRPS-bound peptide, which underpins the unique
316 Cell Chemical Biology 23, March 17, 2016 ª2016 Elsevier Ltd All rights reserved
Gaudelli, N.M., Long, D.H., and Townsend, C.A. (2015). Nature 520, 383–387.
Haslinger, K., Peschke, M., Brieke, C., Maximowitsch, E., and Cryle, M.J. (2015). Nature 521, 105–109. Hur, G.H., Vickery, C.R., and Burkart, M.D. (2012). Nat. Prod. Rep. 29, 1074–1098. Liu, Y., Zheng, T., and Bruner, S.D. (2011). Chem. Biol. 18, 1482–1488. Qiao, C., Wilson, D.J., Bennett, E.M., and Aldrich, C.C. (2007). J. Am. Chem. Soc. 129, 6350–6351. Reimer, J.M., Aloise, M.N., Harrison, P.M., and Schmeing, T.M. (2016). Nature 529, 239–242. Tanovic, A., Samel, S.A., Essen, L.-O., and Marahiel, M.A. (2008). Science 321, 659–663.