PDBsum entry 6hgm

Transport protein

PDB id: 6hgm

Contents

Protein chains

330 a.a.

33 a.a.

Ligands

EDO ×2

Metals

_CL ×3

_CA ×3

Waters ×413

PDB id:

6hgm

Name:

Transport protein

Title:

Crystal structure of alpha1-antichymotrypsin variant newbg-iii-allo: an allosterically controlled new binding globulin with an unprecedentedly high ligand release efficacy

Structure:

Alpha-1-antichymotrypsin. Chain: a. Synonym: act,cell growth-inhibiting gene 24/25 protein,serpin a3. Engineered: yes. Mutation: yes. Other_details: - all residues that are present in the sample sequence but not in the PDB file could not be modelled due to missing electron density - residues following the sequence ..Kitll are part of chain b, as the protein is a family member of serine proteinase inhibitors

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: serpina3, aact, gig24, gig25. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

Resolution:

1.37Å R-factor: 0.169 R-free: 0.181

Authors:

K.Schmidt,Y.A.Muller

Key ref:

B.R.Gardill et al. (2019). NewBG: A surrogate corticosteroid-binding globulin with an unprecedentedly high ligand release efficacy. J Struct Biol, 207, 169-182. PubMed id: 31103428 DOI: 10.1016/j.jsb.2019.05.006

Date:

23-Aug-18 Release date: 29-May-19

Protein chain

P01011  (AACT_HUMAN) -  Alpha-1-antichymotrypsin from Homo sapiens

Seq: 423 a.a.

Struc: 330 a.a.*

Protein chain

P01011  (AACT_HUMAN) -  Alpha-1-antichymotrypsin from Homo sapiens

Seq: 423 a.a.

Struc: 33 a.a.*

* PDB and UniProt seqs differ at 15 residue positions (black crosses)

Enzyme reactions

• Enzyme class: Chains A, B: E.C.?

DOI no: 10.1016/j.jsb.2019.05.006

J Struct Biol 207:169-182 (2019)

PubMed id: 31103428

NewBG: A surrogate corticosteroid-binding globulin with an unprecedentedly high ligand release efficacy.

B.R.Gardill, K.Schmidt, Y.A.Muller.

ABSTRACT

The introduction of ligand-binding sites into proteins and the engineering of molecular allosteric coupling pathways are topical issues in protein design. Here, we show that these issues can be addressed concurrently, using the serpin human α1-antichymotrypsin (ACT) as a model. We have introduced up to 15 amino acid substitutions into ACT, converting it into a surrogate corticosteroid-binding globulin (CBG), thereby creating a new binding globulin (NewBG). Human CBG and ACT share 46% sequence identity, and CBG served as the blue-print for our design, which was guided by side-chain-packing calculations, ITC measurements and crystal structure determinations. Upon transfer of ligand-interacting residues from CBG to ACT and mutation of specific second shell residues, a NewBG variant was obtained, which binds cortisol with 1.5 µM affinity. This novel serpin (NewBG-III) binds cortisol with a 33-fold lower affinity than CBG, but shares a similar ligand-binding profile and binding mode when probed with different steroid ligands and site-directed mutagenesis. An additional substitution, i.e. A349R, created NewBG-III-allo, which introduced an allosteric coupling between ligand binding and the serpin-like S-to-R transition in ACT. In NewBG-III-allo, the proteinase-triggered S-to-R transition leads to a greater than 200-fold reduction in ligand affinity, and crystal structures suggest that this is mediated by the L55V and A349R substitutions. This reduction significantly exceeds the 10-fold reduction in binding affinity observed in human CBG.