How FMN binds to anabaena apoflavodoxin: a hydrophobic encounter at an open binding site.
Molecular recognition begins when two molecules approach and establish interactions of certain strength. The mechanisms of molecular recognition reactions between biological molecules are not well known, and few systems have been analyzed in detail. We investigate here the reaction between an apoprotein and its physiological cofactor (apoflavodoxin and flavin mononucleotide) that binds reversibly to form a non-covalent complex (flavodoxin) involved in electron transfer reactions. We have analyzed the fast binding reactions between the FMN cofactor (and shorter analogs) and wild type (and nine mutant apoflavodoxins where residues interacting with FMN in the final complex have been replaced). The x-ray structures of two such mutants are reported that show the mutations are well tolerated by the protein. From the calculated microscopic binding rate constants we have performed a Phi analysis of the transition state of complex formation that indicates that the binding starts by interaction of the isoalloxazine-fused rings in FMN with residues of its hydrophobic binding site. In contrast, the phosphate in FMN, known to contribute most to the affinity of the final holoflavodoxin complex, is not bound in the transition state complex. Both the effects of ionic strength and of phosphate concentration on the wild type complex rate constant agree with this scenario. As suggested previously by nmr data, it seems that the isoalloxazine-binding site may be substantially open in solution. Interestingly, although FMN is a charged molecule, electrostatic interactions seem not to play a role in directing the binding, unlike what has been reported for other biological complexes. The binding can thus be best described as a hydrophobic encounter at an open binding site.
Lostao
Anabel
A
Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias and Biocomputation and Complex Systems Physics Institute, Universidad de Zaragoza 50009-Zaragoza, Madrid, Spain.
Daoudi
Fatna
F
Irún
Mariá Pilar
MP
Ramon
Alvaro
A
Fernández-Cabrera
Concha
C
Romero
Antonio
A
Sancho
Javier
J
D017033:Anabaena / Q000737:chemistry*; D001059:Apoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D018360:Crystallography, X-Ray; D005486:Flavin Mononucleotide / Q000737:chemistry* / Q000378:metabolism; D005418:Flavodoxin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D009154:Mutation; D009994:Osmolar Concentration; D010710:Phosphates / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation
D001059:Apoproteins; D001426:Bacterial Proteins; D005418:Flavodoxin; D010710:Phosphates; C026932:apoflavodoxin; D005486:Flavin Mononucleotide