Locating high-affinity fatty acid-binding sites on albumin by x-ray crystallography and NMR spectroscopy.
Human serum albumin (HSA) is a versatile transport protein for endogenous compounds and drugs. To evaluate physiologically relevant interactions between ligands for the protein, it is necessary to determine the locations and relative affinities of different ligands for their binding site(s). We present a site-specific investigation of the relative affinities of binding sites on HSA for fatty acids (FA), the primary physiological ligand for the protein. Titration of HSA with [(13)C]carboxyl-labeled FA was used initially to identify three NMR chemical shifts that are associated with high-affinity binding pockets on the protein. To correlate these peaks with FA-binding sites identified from the crystal structures of FA-HSA complexes, HSA mutants were engineered with substitutions of amino acids involved in coordination of the bound FA carboxyl. Titration of [(13)C]palmitate into solutions of HSA mutants for either FA site four (R410A/Y411A) or site five (K525A) within domain III of HSA each revealed loss of a specific NMR peak that was present in spectra of wild-type protein. Because these peaks are among the first three to be observed on titration of HSA with palmitate, sites four and five represent two of the three high-affinity long-chain FA-binding sites on HSA. These assignments were confirmed by titration of [(13)C]palmitate into recombinant domain III of HSA, which contains only sites four and five. These results establish a protocol for direct probing of the relative affinities of FA-binding sites, one that may be extended to examine competition between FA and other ligands for specific binding sites.
Simard
J R
JR
Department of Pharmacology and Experimental Therapeutics, Boston University School of Medicine, MA 02118, USA.
Zunszain
P A
PA
Ha
C-E
CE
Yang
J S
JS
Bhagavan
N V
NV
Petitpas
I
I
Curry
S
S
Hamilton
J A
JA
D001665:Binding Sites; D002247:Carbon Isotopes; D018360:Crystallography, X-Ray; D005227:Fatty Acids / Q000378:metabolism*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D010168:Palmitates; D012709:Serum Albumin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D002247:Carbon Isotopes; D005227:Fatty Acids; D010168:Palmitates; D012709:Serum Albumin