6U18

Directed evolution of a biosensor selective for the macrolide antibiotic clarithromycin

PDB DOI: https://doi.org/10.2210/pdb6U18/pdb

Classification: DNA BINDING PROTEIN/ANTIBIOTIC
Organism(s): Escherichia coli
Expression System: Escherichia coli BL21(DE3)
Mutation(s): Yes

Deposited: 2019-08-15 Released: 2020-08-19
Deposition Author(s): Li, Y., Reed, M., Wright, H.T., Cropp, T.A., Williams, G.
Funding Organization(s): National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)

Experimental Data Snapshot

Method: X-RAY DIFFRACTION
Resolution: 2.00 Å
R-Value Free: 0.221
R-Value Work: 0.202
R-Value Observed: 0.203

Starting Model: experimental
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wwPDB Validation 3D Report Full Report

Ligand Structure Quality Assessment

This is version 1.2 of the entry. See complete history.

Literature

Development of Genetically Encoded Biosensors for Reporting the Methyltransferase-Dependent Biosynthesis of Semisynthetic Macrolide Antibiotics.

Li, Y., Reed, M., Wright, H.T., Cropp, T.A., Williams, G.J.

(2021) ACS Synth Biol

PubMed: 34546703 Search on PubMedSearch on PubMed Central
DOI: https://doi.org/10.1021/acssynbio.1c00151
Primary Citation of Related Structures:
6U18

PubMed Abstract:
Clarithromycin is an improved semisynthetic analogue of the naturally occurring macrolide, erythromycin. The subtle modification of a methyl group on the C-6 hydroxyl group endows the molecule with improved acid stability and results in a clinically useful antibiotic. Here, we show that the effector specificity of the biosensor protein, MphR, can be evolved to selectively recognize clarithromycin and therefore report on the production of this molecule in vivo . In addition, a crystal structure of the evolved variant reveals the molecular basis for selectivity and provides a guide for the evolution of a new metabolic function using this biosensor.

Organizational Affiliation

Department of Chemistry, NC State University, Raleigh, North Carolina 27695, United States.

Macromolecule Content

Total Structure Weight: 44.89 kDa
Atom Count: 3,069
Modelled Residue Count: 367
Deposited Residue Count: 388
Unique protein chains: 1

Macromolecules

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(by identity cutoff) | 3D Structure

Entity ID: 1
Molecule	Chains	Sequence Length	Organism	Details	Image
Erythromycin resistance repressor protein	A, B	194	Escherichia coli	Mutation(s): 4 Gene Names: mphR(A), mphR, A9R57_28010, ACN77_02555, ACN77_03810, ACN77_19745, AKG29_00740, AM465_27045, APT94_14580, B1K96_22845... mphR(A), mphR, A9R57_28010, ACN77_02555, ACN77_03810, ACN77_19745, AKG29_00740, AM465_27045, APT94_14580, B1K96_22845, B9M99_28120, B9N33_26285, B9T59_28650, BANRA_01808, BANRA_05330, BANRA_05395, BE963_00530, BEN53_26070, BET08_23460, BFD68_26430, BJJ90_27670, BK248_28255, BK292_28800, BK334_26705, BK373_25645, BK375_28450, BK383_16415, BK400_25090, BVL39_07785, BVL39_26565, C5N07_27930, C5P01_27745, C5P43_14800, C5P44_28315, C6669_28285, C6986_31345, C7235_25300, C7235_26770, CQP61_00550, CR538_26710, CRT43_27530, CRT46_27035, CWS33_26295, D0X26_00055, D0X26_28400, D3O91_28140, D9N32_01235, DB359_27590, DBY14_07355, DBY14_07475, DBY14_07595, DBY14_07715, DBY14_07835, DBY14_07955, DBY14_08075, DBY14_08195, DBY14_08400, DBY14_09000, DBY14_09120, DBY14_09240, DBY14_09360, DBY14_09480, DBY14_09600, DBY14_09720, DBY14_09840, DBY14_09960, DBY14_10080, DBY14_10200, DBY14_10320, DBY14_10440, DBY14_10560, DBY14_10680, DBY14_10800, DBY14_10920, DBY14_11040, DBY14_11160, DBY14_11280, DL545_01125, DL545_26270, DM102_26995, DM129_26600, DM129_28140, DNQ41_27340, DS732_30515, E4Z89_24290, Eco118UI_26585, ECONIH1_26780, ECTO124_05297, EGT48_01470, EIA21_26670, EJC48_00900, EJC75_00560, EJH97_25820, EPS71_24560, EQ823_24865, EQ830_27025, ERS085366_04056, ERS085404_05054, ERS150873_04751, ETN48_p0081, EVY21_24885, ExPECSC022_02556, ExPECSC036_03428, ExPECSC065_05093, EXX13_27025, EYD11_23970, FORC82_p029, HmCmsJML146_04799, HMPREF3040_01640, MS6198_B124, MS8345_A00020, NCTC13846_04922, U12A_A00163, U14A_A00115, UN86_19865, YDC107_4984
UniProt
Find proteins for Q9EVJ6 (Escherichia coli) Explore Q9EVJ6 Go to UniProtKB: Q9EVJ6
Entity Groups
Sequence Clusters	30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt Group	Q9EVJ6
Sequence Annotations Expand
Reference Sequence

Small Molecules

Ligands 2 Unique
ID	Chains	Name / Formula / InChI Key	2D Diagram	3D Interactions
CTY (Subject of Investigation/LOI) Query on CTY Download Ideal Coordinates CCD File SDF format, chain C [auth A] SDF format, chain E [auth B] MOL2 format, chain C [auth A] MOL2 format, chain E [auth B] mmCIF format, chain C [auth A] mmCIF format, chain E [auth B]	C [auth A], E [auth B]	CLARITHROMYCIN C₃₈ H₆₉ N O₁₃ AGOYDEPGAOXOCK-KCBOHYOISA-N		Interactions Focus chain C [auth A] Focus chain E [auth B] Interactions & Density Focus chain C [auth A] Focus chain E [auth B]
FLC Query on FLC Download Ideal Coordinates CCD File SDF format, chain D [auth A] MOL2 format, chain D [auth A] mmCIF format, chain D [auth A]	D [auth A]	CITRATE ANION C₆ H₅ O₇ KRKNYBCHXYNGOX-UHFFFAOYSA-K		Interactions Focus chain D [auth A] Interactions & Density Focus chain D [auth A]