Loopless Rop: structure and dynamics of an engineered homotetrameric variant of the repressor of primer protein.
The repressor of primer (Rop) protein has become a steady source of surprises concerning the relationship between the sequences and the structures of several of its mutants and variants. Here we add another piece to the puzzle of Rop by showing that an engineered deletion mutant of the protein (corresponding to a deletion of residues 30-34 of the wild-type protein and designed to restore the heptad periodicity at the turn region) results in a complete reorganization of the bundle which is converted from a homodimer to a homotetramer. In contrast (and as previously shown), a two-residue insertion, which also restores the heptad periodicity, is essentially identical with wild-type Rop. The new deletion mutant structure is a canonical, left-handed, all-antiparallel bundle with a completely different hydrophobic core and distinct surface properties. The structure agrees and qualitatively explains the results from functional, thermodynamic, and kinetic studies which indicated that this deletion mutant is a biologically inactive hyperstable homotetramer. Additional insight into the stability and dynamics of the mutant structure has been obtained from extensive molecular dynamics simulations in explicit water and with full treatment of electrostatics.
Glykos
Nicholas M
NM
Department of Molecular Biology and Genetics, Democritus University of Thrace, Dimitras 19, 68100 Alexandroupolis, Greece.
Papanikolau
Yannis
Y
Vlassi
Metaxia
M
Kotsifaki
Dina
D
Cesareni
Giovanni
G
Kokkinidis
Michael
M
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D018360:Crystallography, X-Ray; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid
D001426:Bacterial Proteins; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; C054243:Rop protein, ColE1 plasmid