PDBsum entry 4j9t

Structural genomics, unknown function

PDB id: 4j9t

Contents

Protein chain

355 a.a.

Ligands

GOL

ARS

Waters ×527

PDB id:

4j9t

Name:

Structural genomics, unknown function

Title:

Crystal structure of a putative, de novo designed unnatural amino acid dependent metalloprotein, northeast structural genomics consortium target or61

Structure:

Designed unnatural amino acid dependent metalloprotein. Chain: a. Synonym: neuraminidase, sialidase. Engineered: yes. Mutation: yes

Source:

Micromonospora viridifaciens. Organism_taxid: 1881. Gene: neda. Expressed in: escherichia coli. Expression_system_taxid: 469008.

Resolution:

1.40Å R-factor: 0.138 R-free: 0.166

Authors:

F.Forouhar,S.Lew,J.Seetharaman,J.H.Mills,S.D.Khare,J.K.Everett, D.Baker,G.T.Montelione,J.F.Hunt,L.Tong,Northeast Structural Genomics Consortium (Nesg)

Key ref:

J.H.Mills et al. (2013). Computational design of an unnatural amino acid dependent metalloprotein with atomic level accuracy. J Am Chem Soc, 135, 13393-13399. PubMed id: 23924187 DOI: 10.1021/ja403503m

Date:

17-Feb-13 Release date: 27-Mar-13

Protein chain

Q02834  (NANH_MICVI) -  Sialidase from Micromonospora viridifaciens

Seq: 647 a.a.

Struc: 355 a.a.*

* PDB and UniProt seqs differ at 12 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.3.2.1.18 - exo-alpha-sialidase.
• Reaction: Hydrolysis of alpha-(2->3)-, alpha-(2->6)-, alpha-(2->8)-glycosidic linkages of terminal sialic residues in oligosaccharides, glycoproteins, glycolipids, colominic acid and synthetic substrates.

DOI no: 10.1021/ja403503m

J Am Chem Soc 135:13393-13399 (2013)

PubMed id: 23924187

Computational design of an unnatural amino acid dependent metalloprotein with atomic level accuracy.

J.H.Mills, S.D.Khare, J.M.Bolduc, F.Forouhar, V.K.Mulligan, S.Lew, J.Seetharaman, L.Tong, B.L.Stoddard, D.Baker.

ABSTRACT

Genetically encoded unnatural amino acids could facilitate the design of proteins and enzymes of novel function, but correctly specifying sites of incorporation and the identities and orientations of surrounding residues represents a formidable challenge. Computational design methods have been used to identify optimal locations for functional sites in proteins and design the surrounding residues but have not incorporated unnatural amino acids in this process. We extended the Rosetta design methodology to design metalloproteins in which the amino acid (2,2'-bipyridin-5yl)alanine (Bpy-Ala) is a primary ligand of a bound metal ion. Following initial results that indicated the importance of buttressing the Bpy-Ala amino acid, we designed a buried metal binding site with octahedral coordination geometry consisting of Bpy-Ala, two protein-based metal ligands, and two metal-bound water molecules. Experimental characterization revealed a Bpy-Ala-mediated metalloprotein with the ability to bind divalent cations including Co(2+), Zn(2+), Fe(2+), and Ni(2+), with a Kd for Zn(2+) of ∼40 pM. X-ray crystal structures of the designed protein bound to Co(2+) and Ni(2+) have RMSDs to the design model of 0.9 and 1.0 Å respectively over all atoms in the binding site.