NDLI: Structure of an Engineered β-Lactamase Maltose Binding Protein Fusion Protein: Insights into Heterotropic Allosteric Regulation

Content Provider	PubMed Central
Author	Ke, Wei Laurent, Abigail H. Armstrong, Morgan D. Chen, Yuchao Smith, William E. Liang, Jing Wright, Chapman M. Ostermeier, Marc Akker, Focco Van Den
Editor	Kobe, Bostjan
Copyright Year	2012
Abstract	Engineering novel allostery into existing proteins is a challenging endeavor to obtain novel sensors, therapeutic proteins, or modulate metabolic and cellular processes. The RG13 protein achieves such allostery by inserting a circularly permuted TEM-1 β-lactamase gene into the maltose binding protein (MBP). RG13 is positively regulated by maltose yet is, serendipitously, inhibited by Zn2+ at low µM concentration. To probe the structure and allostery of RG13, we crystallized RG13 in the presence of mM Zn2+ concentration and determined its structure. The structure reveals that the MBP and TEM-1 domains are in close proximity connected via two linkers and a zinc ion bridging both domains. By bridging both TEM-1 and MBP, Zn2+ acts to “twist tie” the linkers thereby partially dislodging a linker between the two domains from its original catalytically productive position in TEM-1. This linker 1 contains residues normally part of the TEM-1 active site including the critical β3 and β4 strands important for activity. Mutagenesis of residues comprising the crystallographically observed Zn2+ site only slightly affected Zn2+ inhibition 2- to 4-fold. Combined with previous mutagenesis results we therefore hypothesize the presence of two or more inter-domain mutually exclusive inhibitory Zn2+ sites. Mutagenesis and molecular modeling of an intact TEM-1 domain near MBP within the RG13 framework indicated a close surface proximity of the two domains with maltose switching being critically dependent on MBP linker anchoring residues and linker length. Structural analysis indicated that the linker attachment sites on MBP are at a site that, upon maltose binding, harbors both the largest local Cα distance changes and displays surface curvature changes, from concave to relatively flat becoming thus less sterically intrusive. Maltose activation and zinc inhibition of RG13 are hypothesized to have opposite effects on productive relaxation of the TEM-1 β3 linker region via steric and/or linker juxtapositioning mechanisms.
Related Links	http://dx.doi.org/10.1371/journal.pone.0039168
Starting Page	39168
File Format	PDF
ISSN	19326203
e-ISSN	19326203
Journal	PLoS ONE
Issue Number	6
Volume Number	7
Language	English
Publisher	Public Library of Science
Publisher Date	2012-06-01
Access Restriction	Open
Rights Holder	Public Library of Science
Subject Keyword	Biochemistry, Genetics and Molecular Biology(all) Agricultural and Biological Sciences(all) Medicine(all) Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Multidisciplinary

Sl.	Authority	Responsibilities	Communication Details
1	Ministry of Education (GoI), Department of Higher Education	Sanctioning Authority	https://www.education.gov.in/ict-initiatives
2	Indian Institute of Technology Kharagpur	Host Institute of the Project: The host institute of the project is responsible for providing infrastructure support and hosting the project	https://www.iitkgp.ac.in
3	National Digital Library of India Office, Indian Institute of Technology Kharagpur	The administrative and infrastructural headquarters of the project	Dr. B. Sutradhar bsutra@ndl.gov.in
4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
5	Website/Portal (Helpdesk)	Queries regarding NDLI and its services	support@ndl.gov.in
6	Contents and Copyright Issues	Queries related to content curation and copyright issues	content@ndl.gov.in
7	National Digital Library of India Club (NDLI Club)	Queries related to NDLI Club formation, support, user awareness program, seminar/symposium, collaboration, social media, promotion, and outreach	clubsupport@ndl.gov.in
8	Digital Preservation Centre (DPC)	Assistance with digitizing and archiving copyright-free printed books	dpc@ndl.gov.in
9	IDR Setup or Support	Queries related to establishment and support of Institutional Digital Repository (IDR) and IDR workshops	idr@ndl.gov.in

NMR characterization of an engineered domain fusion between maltose binding protein and TEM1 beta-lactamase provides insight into its structure and allosteric mechanism.

A Maltose-Binding Protein Fusion Construct Yields a Robust Crystallography Platform for MCL1

Identification of Residues in β-Lactamase Critical for Binding β-Lactamase Inhibitory Protein

Prokaryotic Soluble Overexpression and Purification of Human VEGF165 by Fusion to a Maltose Binding Protein Tag

Prokaryotic Soluble Overexpression and Purification of Bioactive Human Growth Hormone by Fusion to Thioredoxin, Maltose Binding Protein, and Protein Disulfide Isomerase

Maltose-Binding Protein Enhances Secretion of Recombinant Human Granzyme B Accompanied by In Vivo Processing of a Precursor MBP Fusion Protein

Anthrax Toxin Uptake by Primary Immune Cells as Determined with a Lethal Factor-β-Lactamase Fusion Protein

Crystal Structure of DIM-1, an Acquired Subclass B1 Metallo-β-Lactamase from Pseudomonas stutzeri

Maltose-Binding Protein (MBP), a Secretion-Enhancing Tag for Mammalian Protein Expression Systems

Structure of an Engineered β-Lactamase Maltose Binding Protein Fusion Protein: Insights into Heterotropic Allosteric Regulation

Similar Documents

NMR characterization of an engineered domain fusion between maltose binding protein and TEM1 beta-lactamase provides insight into its structure and allosteric mechanism.

A Maltose-Binding Protein Fusion Construct Yields a Robust Crystallography Platform for MCL1

Identification of Residues in β-Lactamase Critical for Binding β-Lactamase Inhibitory Protein

Prokaryotic Soluble Overexpression and Purification of Human VEGF165 by Fusion to a Maltose Binding Protein Tag

Prokaryotic Soluble Overexpression and Purification of Bioactive Human Growth Hormone by Fusion to Thioredoxin, Maltose Binding Protein, and Protein Disulfide Isomerase

Maltose-Binding Protein Enhances Secretion of Recombinant Human Granzyme B Accompanied by In Vivo Processing of a Precursor MBP Fusion Protein

Anthrax Toxin Uptake by Primary Immune Cells as Determined with a Lethal Factor-β-Lactamase Fusion Protein

Crystal Structure of DIM-1, an Acquired Subclass B1 Metallo-β-Lactamase from Pseudomonas stutzeri

Maltose-Binding Protein (MBP), a Secretion-Enhancing Tag for Mammalian Protein Expression Systems

Structure of an Engineered β-Lactamase Maltose Binding Protein Fusion Protein: Insights into Heterotropic Allosteric Regulation