Structure of a domain-opened mutant (R121D) of the human lactoferrin N-lobe refined from a merohedrally twinned crystal form.
Human lactoferrin is an iron-binding protein with a bilobal structure. Each lobe contains a high-affinity binding site for a single Fe(3+) ion and an associated CO(3)(2-) ion. Although iron binds very tightly, it can be released at low pH, with an accompanying conformational change in which the two domains move apart. The Arg121Asp (R121D) mutant of the N-lobe half-molecule of human lactoferrin was constructed in order to test whether the Asp121 side chain could substitute for the CO(3)(2-) ion at the iron-binding site. The R121D mutant protein was crystallized in its apo form as it lost iron during crystallization. The crystals were also merohedrally twinned, with a twin fraction close to 0.5. Starting from the initial molecular-replacement solution [Breyer et al. (1999), Acta Cryst. D55, 129-138], the structure has been refined at 3.0 A resolution to an R factor of 13.9% (R(free) of 19.9%). Despite the moderate resolution, the high solvent content and non-crystallographic symmetry contributed to electron-density maps of excellent quality. Weakened iron binding by the R121D mutant is explained by occlusion of the anion-binding site by the Asp side chain. The opening of the two domains in the apoR121D structure (a rotation of 54 degrees ) closely matches that of the N-lobe in full-length lactoferrin, showing that the extent of the conformational change depends on properties inherent to the N-lobe. Differences in the C-terminal portion of the N-lobe (residues 321-332) for apoR121D relative to the closed wild-type iron-bound structure point to the importance of this region in stabilizing the open form.
Jameson
Geoffrey B
GB
Department of Chemistry and Biochemistry, Massey University, Palmerston North, New Zealand.
Anderson
Bryan F
BF
Breyer
Wendy A
WA
Day
Catherine L
CL
Tweedie
John W
JW
Baker
Edward N
EN
D019943:Amino Acid Substitution; D001120:Arginine / Q000737:chemistry / Q000235:genetics; D001224:Aspartic Acid / Q000737:chemistry / Q000235:genetics; D003460:Crystallization; D018360:Crystallography, X-Ray; D006801:Humans; D007781:Lactoferrin / Q000737:chemistry*; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D014168:Transferrin / Q000737:chemistry
D014168:Transferrin; D001224:Aspartic Acid; D001120:Arginine; D007781:Lactoferrin