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The X-ray structure of a recombinant major urinary protein at 1.75 A resolution. A comparative study of X-ray and
P R Kuser1, L Franzoni, E Ferrari
1Laboratório Nacional de Luz Síncrotron, Caixa Postal 6192, CEP 13084-971 Campinas, SP, Brazil.
Abstract:
Major urinary proteins belong to the lipocalin family and are present in the urine of rodents as an ensemble of isoforms with pheromonal activity. The crystal structure of a recombinant mouse MUP (rMUP) was solved by the molecular-replacement technique and refined to an R factor and R(free) of 20 and 26.5%, respectively, at 1.75 A resolution. The structure was compared with an NMR model and with a crystallographic structure of the wild-type form of the protein. The crystal structures determined in different space groups present significantly smaller conformational differences amongst themselves than in comparison with NMR models. Some, but not all, of the conformational differences between the crystal and solution structures can be explained by the influence of crystallographic contacts. Most of the differences between the NMR and X-ray structures were found in the N-terminus and loop regions. A number of side chains lining the hydrophobic pocket of the molecule are more tightly packed in the NMR structure than in the crystallographic model. Surprisingly, clear and continuous electron density for a ligand was observed inside the hydrophobic pocket of this recombinant protein. Conformation of the ligand modelled inside the density is coherent with the results of recent NMR experiments.
Insights
The crystal structure of recombinant mouse major urinary protein (rMUP) reveals its 3D conformation and a bound ligand. This provides insights into lipocalin protein structures and pheromonal activity in rodents.
Area of Science:
- Structural biology
- Biochemistry
- Molecular biology
Background:
- Major urinary proteins (MUPs) are lipocalins found in rodent urine.
- These proteins possess pheromonal activity and exist as multiple isoforms.
Purpose of the Study:
- To determine the crystal structure of recombinant mouse major urinary protein (rMUP).
- To compare the crystal structure with NMR and wild-type structures.
- To investigate conformational differences and ligand binding.
Main Methods:
- Molecular replacement technique for crystal structure determination.
- X-ray crystallography at 1.75 Å resolution.
- Comparison with Nuclear Magnetic Resonance (NMR) models.
Main Results:
- The crystal structure of rMUP was refined to high resolution.
- Crystal structures showed minor conformational differences compared to each other.
- Significant differences were observed between crystal and NMR structures, particularly in loop regions.
- A ligand was unexpectedly found within the hydrophobic pocket of rMUP.
Conclusions:
- Crystallographic contacts influence protein conformation in crystal structures.
- NMR and X-ray structures differ mainly in flexible regions.
- The identified ligand's conformation aligns with NMR experimental data, suggesting its biological relevance.