Prediction of structural and functional relationships of Repeat 1 of human interphotoreceptor retinoid-binding protein (IRBP) with other proteins.
OBJECTIVE
We compared the structure and function of interphotoreceptor retinoid-binding protein (IRBP) related proteins and predicted domain and secondary structure within each repeat of IRBP and its relatives. We tested whether tail specific protease (Tsp), which bears sequence similarity to IRBP Domain B, binds fatty acids or retinoids, and whether IRBP possessed protease activity resembling Tsp's catalytic function. These tests helped us to learn whether the primary sequence similarities of family members extended to higher order structural and functional levels.


METHODS
Predictions derived from multiple sequence alignments among IRBP and Tsp family members and secondary structure computer programs were carried out. The first repeat of human IRBP (EcR1) and Tsp were expressed, purified, and tested for binding properties. Tsp was examined for fluorescence enhancement of retinol or 16-anthroyloxy-palmitic acid (16-AP) to test for ligand binding. IRBP was tested for protease activity.


RESULTS
Tsp did not exhibit fluorescence enhancement with retinol or 16-AP. IRBP did not exhibit protease activity. The positions of critical residues needed for the ligand binding properties of retinol were predicted. Primary sequence and three-dimensional similarity was found between Domain A of IRBP Repeat 3 and eglin c.


CONCLUSIONS
The sequence similarity of Tsp and IRBP raised the possibility that each might share the function of the other protein: IRBP might possess protease activity or Tsp might possess retinoid or fatty acid binding activity. Our studies do not support such a shared function hypothesis, and suggest that the sequence similarity is the result of maintenance of structure. The finding of similarity to eglin c in Domain A suggests the possibility of a tight interaction between Domain A and Domain B, possibly implying the need for Domain A in retinoid-binding, and suggesting that both Domains should be present in testing mutations. The positions of predicted critical amino acids suggest models in which a large binding pocket holds the retinoid or fatty acid ligand. These predictions are tested in a companion paper.
Gross
E A
EA
Department of Ophthalmology, Emory University, Atlanta, GA 30322, USA.
Li
G R
GR
Lin
Z Y
ZY
Ruuska
S E
SE
Boatright
J H
JH
Mian
I S
IS
Nickerson
J M
JM
D016000:Cluster Analysis; D010450:Endopeptidases / Q000737:chemistry*; D005136:Eye Proteins; D006801:Humans; D008024:Ligands; D008390:Markov Chains; D010169:Palmitic Acids / Q000737:chemistry; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011506:Proteins; D012177:Retinol-Binding Proteins / Q000737:chemistry*; D016415:Sequence Alignment; D015843:Serpins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D014801:Vitamin A / Q000737:chemistry; D000099214:Retinol-Binding Proteins, Interstitial
D010450:Endopeptidases; D005136:Eye Proteins; D008024:Ligands; D010169:Palmitic Acids; D011506:Proteins; D012177:Retinol-Binding Proteins; D015843:Serpins; D014801:Vitamin A; D000099214:Retinol-Binding Proteins, Interstitial; C028656:eglin proteinase inhibitors; C047265:16-(9-anthroyloxy)palmitic acid; C072135:C-terminal processing peptidase