Construction and characterization of a radio-iodinatable mutant of recombinant human CD4.
Recombinant soluble human CD4 (rsCD4) has been used in iodinated form to study the interaction of CD4 with its ligands. However, the utility of [125I]-rsCD4 is limited because rsCD4 is inefficiently iodinated and the iodinated protein is poorly active. The iodination properties of rsCD4 most likely reflect the poor accessibility of the tyrosine residues, apparent from the available X-ray structures. We have generated an iodinatable mutant of rsCD4 by substituting Tyr for Phe(179) in the flexible, solvent-exposed C-terminal region of rsCD4(183), a truncated form of CD4 that consists of the first 183 residues of CD4 and includes the binding sites for HIV-1 gp120 and MHC class II molecules. When F179Y rsCD4(183) is iodinated under trace-labeling conditions, the efficiency of 125I incorporation and the percentage of iodinated molecules that are active are much enhanced compared with WT rsCD4. Moreover, trace-labeled [125I]-F179Y rsCD4(183) has the same affinity for HIV-1 rgp120 as unlabeled WT rsCD4. The improved activity of trace-labeled [125I]-F179Y rsCD4(183) appears to be due to effective competition by Y179 for reactive iodine species that, in WT rsCD4, react with traces of denatured protein and/or with residues critical for activity or conformational integrity. The incorporation of accessible tyrosine residues may improve the iodinatibility of a protein both by introducing a readily iodinatable residue and by protecting sensitive proteins from adverse reactions.
Ferrer
M
M
Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA.
Godbout
K L
KL
Sullivan
B J
BJ
Austen
D A
DA
Sanderson
C T
CT
Kelley
K C
KC
Osburne
M S
MS
Harrison
S C
SC
van Schravendijk
M R
MR
D000818:Animals; D015704:CD4 Antigens / Q000378:metabolism*; D015699:HIV Envelope Protein gp120 / Q000378:metabolism; D006801:Humans; D007457:Iodine Radioisotopes; D007553:Isotope Labeling; D051379:Mice; D009154:Mutation; D011994:Recombinant Proteins / Q000378:metabolism
D015704:CD4 Antigens; D015699:HIV Envelope Protein gp120; D007457:Iodine Radioisotopes; D011994:Recombinant Proteins