Role of conserved residues in structure and stability: tryptophans of human serum retinol-binding protein, a model for the lipocalin superfamily.


Abstract

Serum retinol binding protein (RBP) is a member of the lipocalin family, proteins with up-and-down beta-barrel folds, low levels of sequence identity, and diverse functions. Although tryptophan 24 of RBP is highly conserved among lipocalins, it does not play a direct role in activity. To determine if Trp24 and other conserved residues have roles in stability and/or folding, we investigated the effects of conservative substitutions for the four tryptophans and some adjacent residues on the structure, stability, and spectroscopic properties of apo-RBP. Crystal structures of recombinant human apo-RBP and of a mutant with substitutions for tryptophans 67 and 91 at 1.7 A and 2.0 A resolution, respectively, as well as stability measurements, indicate that these relatively exposed tryptophans have little influence on structure or stability. Although Trp105 is largely buried in the wall of the beta-barrel, it can be replaced with minor effects on stability to thermal and chemical unfolding. In contrast, substitutions of three different amino acids for Trp24 or replacement of Arg139, a conserved residue that interacts with Trp24, lead to similar large losses in stability and lower yields of native protein generated by in vitro folding. The results and the coordinated nature of natural substitutions at these sites support the idea that conserved residues in functionally divergent homologs have roles in stabilizing the native relative to misfolded structures. They also establish conditions for studies of the kinetics of folding and unfolding by identifying spectroscopic signals for monitoring the formation of different substructures. Study holds ProTherm entries: 12068, 12069, 12070, 12071, 12072, 12073, 12074, 12075, 12076, 12077, 12078 Extra Details: protein stability; mutagenesis; evolution; conserved residues; lipocalins;,protein folding; serum retinol-binding protein; crystal structures

Submission Details

ID: ikoDyUuB4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:43 p.m.

Version: 1

Publication Details
Greene LH;Chrysina ED;Irons LI;Papageorgiou AC;Acharya KR;Brew K,Protein Sci. (2001) Role of conserved residues in structure and stability: tryptophans of human serum retinol-binding protein, a model for the lipocalin superfamily. PMID:11604536
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1KT6 2003-06-03 1.1 Crystal structure of bovine holo-RBP at pH 9.0
1KT7 2003-06-03 1.27 Crystal structure of bovine holo-RBP at pH 7.0
1KT3 2003-06-03 1.4 Crystal structure of bovine holo-RBP at pH 2.0
1KT5 2003-06-03 1.46 Crystal structure of bovine holo-RBP at pH 4.0
1KT4 2003-06-03 1.46 Crystal structure of bovine holo-RBP at pH 3.0
5NU9 2018-03-07 1.5 Structure of human amniotic fluid RBP4 saturated with palmitate
5NU2 2018-03-07 1.5 Structure of non-fluorescent human urine RBP4
5NU7 2018-03-07 1.5 Structure of human holo plasma RBP4
5NU8 2018-03-07 1.59 Structure of human urine RBP4 saturated with palmitate
5NUB 2018-03-14 1.6 Structure of human amniotic fluid RBP4 saturated with laurate
5NUA 2018-03-07 1.6 Structure of human urine RBP4 saturated with laurate
1AQB 1998-01-28 1.65 RETINOL-BINDING PROTEIN (RBP) FROM PIG PLASMA
2WQ9 2010-09-01 1.65 Crystal Structure of RBP4 bound to Oleic Acid
5NU6 2018-03-07 1.68 Structure of non-fluorescent human amniotic fluid RBP4
1JYD 2003-07-01 1.7 Crystal Structure of Recombinant Human Serum Retinol-Binding Protein at 1.7 A Resolution
1HBQ 1994-01-31 1.7 CRYSTAL STRUCTURE OF LIGANDED AND UNLIGANDED FORMS OF BOVINE PLASMA RETINOL-BINDING PROTEIN
1FEL 1994-11-01 1.8 CRYSTALLOGRAPHIC STUDIES ON COMPLEXES BETWEEN RETINOIDS AND PLASMA RETINOL-BINDING PROTEIN
2WR6 2010-09-15 1.8 Structure of the complex of RBP4 with linoleic acid
1HBP 1994-01-31 1.9 CRYSTAL STRUCTURE OF LIGANDED AND UNLIGANDED FORMS OF BOVINE PLASMA RETINOL-BINDING PROTEIN
1ERB 1994-01-31 1.9 THE INTERACTION OF N-ETHYL RETINAMIDE WITH PLASMA RETINOL-BINDING PROTEIN (RBP) AND THE CRYSTAL STRUCTURE OF THE RETINOID-RBP COMPLEX AT 1.9 ANGSTROMS RESOLUTION
1FEN 1994-11-01 1.9 CRYSTALLOGRAPHIC STUDIES ON COMPLEXES BETWEEN RETINOIDS AND PLASMA RETINOL-BINDING PROTEIN
1FEM 1994-11-01 1.9 CRYSTALLOGRAPHIC STUDIES ON COMPLEXES BETWEEN RETINOIDS AND PLASMA RETINOL-BINDING PROTEIN
1JYJ 2003-07-01 2.0 Crystal Structure of a Double Variant (W67L/W91H) of Recombinant Human Serum Retinol-binding Protein at 2.0 A Resolution
5NTY 2018-03-07 2.0 Structure of non-fluorescent human plasma RBP4
1RBP 1991-07-15 2.0 CRYSTALLOGRAPHIC REFINEMENT OF HUMAN SERUM RETINOL BINDING PROTEIN AT 2 ANGSTROMS RESOLUTION
4O9S 2014-07-02 2.3 Crystal structure of Retinol-Binding Protein 4 (RBP4)in complex with a non-retinoid ligand
4PSQ 2014-07-02 2.4 Crystal Structure of Retinol-Binding Protein 4 (RBP4) in complex with a non-retinoid ligand
1BRQ 1994-01-31 2.5 CRYSTAL STRUCTURE OF THE TRIGONAL FORM OF HUMAN PLASMA RETINOL-BINDING PROTEIN AT 2.5 ANGSTROMS RESOLUTION
1BRP 1994-01-31 2.5 CRYSTAL STRUCTURE OF THE TRIGONAL FORM OF HUMAN PLASMA RETINOL-BINDING PROTEIN AT 2.5 ANGSTROMS RESOLUTION
2WQA 2010-09-01 2.85 Complex of TTR and RBP4 and Oleic Acid
3FMZ 2009-01-27 2.9 Crystal Structure of Retinol-Binding Protein 4 (RBP4) in complex with non-retinoid ligand
1RLB 1996-04-11 3.1 RETINOL BINDING PROTEIN COMPLEXED WITH TRANSTHYRETIN
1QAB 1999-02-16 3.2 The structure of human retinol binding protein with its carrier protein transthyretin reveals interaction with the carboxy terminus of RBP
3BSZ 2008-11-11 3.38 Crystal structure of the transthyretin-retinol binding protein-Fab complex

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
91.8 Retinol-binding protein 4 P06912 RET4_RABIT
92.3 Retinol-binding protein 4 P18902 RET4_BOVIN
92.9 Retinol-binding protein 4 P27485 RET4_PIG
94.5 Retinol-binding protein 4 Q28369 RET4_HORSE
94.5 Retinol-binding protein 4 M5AXY1 RET4_FELCA
100.0 Retinol-binding protein 4 P61641 RET4_PANTR
100.0 Retinol-binding protein 4 P02753 RET4_HUMAN