PDBsum entry 1acd

Fatty acid binding protein

PDB id: 1acd

Contents

Protein chain

131 a.a. *

* Residue conservation analysis

PDB id:

1acd

Name:

Fatty acid binding protein

Title:

V32d/f57h mutant of murine adipocyte lipid binding protein

Structure:

Adipocyte lipid binding protein. Chain: a. Synonym: albp. Engineered: yes. Mutation: yes

Source:

Mus musculus. House mouse. Organism_taxid: 10090. Cell: adipocyte. Expressed in: escherichia coli. Expression_system_taxid: 562

Resolution:

2.70Å R-factor: 0.198 R-free: 0.276

Authors:

J.Ory,C.D.Kane,M.Simpson,L.J.Banaszak,D.A.Bernlohr

Key ref:

J.Ory et al. (1997). Biochemical and crystallographic analyses of a portal mutant of the adipocyte lipid-binding protein. J Biol Chem, 272, 9793-9801. PubMed id: 9092513 DOI: 10.1074/jbc.272.15.9793

Date:

06-Feb-97 Release date: 16-Jun-97

Protein chain

P04117  (FABP4_MOUSE) -  Fatty acid-binding protein, adipocyte from Mus musculus

Seq: 132 a.a.

Struc: 131 a.a.*

* PDB and UniProt seqs differ at 4 residue positions (black crosses)

DOI no: 10.1074/jbc.272.15.9793

J Biol Chem 272:9793-9801 (1997)

PubMed id: 9092513

Biochemical and crystallographic analyses of a portal mutant of the adipocyte lipid-binding protein.

J.Ory, C.D.Kane, M.A.Simpson, L.J.Banaszak, D.A.Bernlohr.

ABSTRACT

A number of crystallographic studies of the adipocyte lipid-binding protein have established that the fatty acid-binding site is within an internalized water-filled cavity. The same studies have also suggested the existence of a region physically distinct from the fatty acid-binding site which connects the cavity of the protein with the external solvent, hereafter referred to as the portal. In an effort to examine the portal region, we have used site-directed mutagenesis to introduce the mutations V32D/F57H into the murine ALBP cDNA. Mutant protein has been isolated, crystallized, and its stability and binding properties studied by biochemical methods. As assessed by guanidine-HCl denaturation, the mutant form exhibited a slight overall destabilization relative to the wild-type protein under both acid and alkaline conditions. Accessibility to the cavity in both the mutant and wild-type proteins was observed by stopped-flow analysis of the modification of a cavity residue, Cys117, by the sulfhydryl reactive agent 5, 5'-dithiobis(2-nitrobenzoic acid) at pH 8.5. Cys117 of V32D/F57H ALBP was modified 7-fold faster than the wild-type protein. The ligand binding properties of both the V32D/F57H mutant and wild-type proteins were analyzed using a fluorescent probe at pH 6.0 and 8.0. The apparent dissociation constants for 1-anilinonaphthalene-8-sulfonic acid were approximately 9-10-fold greater than the wild-type protein, independent of pH. In addition, there is a 6-fold increase in the Kd for oleic acid for the portal mutant relative to the wild-type at pH 8.0. To study the effect of pH on the double mutant, it was crystallized and analyzed in two distinct space groups at pH 4.5 and 6.4. While in general the differences in the overall main chain conformations are negligible, changes were observed in the crystallographic structures near the site of the mutations. At both pH values, the mutant side chains are positioned somewhat differently than in wild-type protein. To ensure that the mutations had not altered ionic conditions near the binding site, the crystallographic coordinates were used to monitor the electrostatic potentials from the head group site to the positions near the portal region. The differences in the electrostatic potentials were small in all regions, and did not explain the differences in ligand affinity. We present these results within the context of fatty acid binding and suggest lipid association is more complex than that described within a single equilibrium event.

Selected figure(s)

Figure 4.
Fig. 4. The crystal structures of the V32D/F57H mutant near the portal region. The stereodiagram contains a ball and stick representation of the crystal structures of the V32D/F57H mutant near the expected portal region of ALBP. Notice His57 is hydrogen bonded to Ser55 in the pH 4.5 form, but makes a bond with Asp32 in the pH 6.4 form. The methyl end of HDS is shown as well, illustrating the residues close proximity to the ligand. HDS binding was modeled^ after the V32D/F57H mutant coordinates were aligned with the crystal structure of ALBP containing bound HDS.

Figure 6.
Fig. 6. Schematic representation of the open and closed conformations of ALBP. Schematic representation of the apo- and holoprotein forms of ALBP in their open and closed conformations. Alternation of the ligand conformation between the open and closed conformations is speculative.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (1997, 272, 9793-9801) copyright 1997.

Figures were selected by an automated process.