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. 2005 Nov 15;102(46):16830-5.
doi: 10.1073/pnas.0504838102. Epub 2005 Nov 7.

Structure and activity of the axon guidance protein MICAL

Mythili Nadella  1 , Mario A Bianchet, Sandra B Gabelli, Jennifer Barrila, L Mario Amzel
Affiliations

Structure and activity of the axon guidance protein MICAL

Mythili Nadella et al. Proc Natl Acad Sci U S A. .

Abstract

During development, neurons are guided to their targets by short- and long-range attractive and repulsive cues. MICAL, a large multidomain protein, is required for the combined action of semaphorins and plexins in axon guidance. Here, we present the structure of the N-terminal region of MICAL (MICAL(fd)) determined by x-ray diffraction to 2.0 A resolution. The structure shows that MICAL(fd) is an FAD-containing module structurally similar to aromatic hydroxylases and amine oxidases. In addition, we present biochemical data that show that MICAL(fd) is a flavoenzyme that in the presence of NADPH reduces molecular oxygen to H(2)O(2) (K(m,NAPDH) = 222 microM; k(cat) = 77 sec(-1)), a molecule with known signaling properties. We propose that the H(2)O(2) produced by this reaction may be one of the signaling molecules involved in axon guidance by MICAL.

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Figures

Fig. 1.
Fig. 1.
Secondary and tertiary structure of MICALfd.(A) Ribbon diagram of the MICAL FAD-binding domain. In this view, the two subdomains of the structure and the two strands that connect them are clearly visible. In domain-1 (large domain at the top of the figure), the central core is formed by a parallel β-sheet. All of the connecting helices are in one side of the sheet, and a short β-hairpin covers the other side. Domain-2 (small domain at the bottom) contains an antiparallel β-sheet and several short helices. (B) Schematic representation of the MICALfd secondary structure. Strands are represented by arrows, helices are represented by cylinders, and loops are represented by a connecting line. Residue numbers at the beginning and the end of elements of secondary structure are shown. The different structural features are colored for identification. The N-terminal portion, containing the two helical hairpins, is colored blue. Other elements of domain-1 are colored red, maroon, and orange (red, main β-sheet; maroon, helices at one side; orange, strands at the other side). Domain-2 is colored light green, and the two connecting strands are colored pale green.
Fig. 2.
Fig. 2.
Alignment of the sequences of MICALfd with structural homologs. Single-letter amino acid codes are used. Three sequences are included. (i) MICAL: residues 85-442 of the MICAL-1 of mouse. Residues 1-484 of this protein correspond to MICALfd, but residues 1-84 of MICALfd form a domain that is not present in the other enzymes. (ii) Human monoamine oxidase B (Protein Data Bank entry 1GOS). Only residues 4-95 and 198-456 are shown. (iii) pHBH from Pseudomonas fluorescens residues 1-340 (Protein Data Bank entry 1PBE). The positions of the elements of secondary structure of MICAL, pHBH, and 1GOS are shown at the top of each sequence. The sequences corresponding to the conserved motifs are shown with blue letters. Letters with green background correspond to regions with large insertions and deletions. The two alanine residues with the green background correspond to the mutations introduced to improve the crystals. Residues identical in all three sequences have magenta backgrounds. Residues that contact the FAD cofactor are shown with cyan background. An insertion of 100 residues between α7 and β4, present only in amine oxidase, has been omitted.
Fig. 3.
Fig. 3.
Analytical ultracentrifugation experiments. The sedimentation of MICALfd was analyzed with a Beckman Coulter XL-I analytical ultracentrifuge at 20°C and 4°C and at 50,000 rpm. Only the experiments at 20°C are shown. (A) Typical sedimentation velocity absorbance trace of MICALfd at 370 nm. (B) Residuals of the experimental fits. (C) Continuous sedimentation coefficient distribution of the protein. (D) Continuous molar mass distribution of the protein showing a single peak with a molecular mass of 52.4 kDa.
Fig. 4.
Fig. 4.
Binding of FAD to MICALfd.(A) Electron density of the bound FAD. The SigmaA-weighted map was calculated by using 2mFo-DFc coefficients (45). Some of the protein residues contacting the FAD are shown. (B) Stereoview of the interactions of FAD with MICALfd residues. The following coloring scheme was used for the protein atoms: C, cyan; N, blue; O, red. Water molecules are represented by red spheres. Colors used for the FAD differ only in that carbons are green and phosphates are magenta. (C) Two bound conformations of FAD. The surface of the protein in the cavity around the FAD is shown as an electrostatic surface. The protein atoms surrounding the cavity are shown with carbons colored green, nitrogens colored blue, and oxygens colored orange. The same colors are used for the FAD in the “out” conformation (crystal) with the addition of magenta for phosphorous. The portion of the FAD that has moved in the “in” conformation (model) has yellow carbon atoms.
Fig. 5.
Fig. 5.
Kinetic characterization of the MICALfd reaction. (A) Kinetics of NADPH oxidation and H2O2 production. MICALfd (6.02 nM) was incubated in a reaction mixture containing 200 μM NADPH and 3 units of the Amplex Red peroxide/peroxidase assay kit with 1.5 mM 10-acetyl-3,7-dihydroxyphenoxazine. The absorbance peak at 340 nm reflects the concentration of reduced NADPH, and the peak at 560 nm reflects the concentration of H2O2. The experiment ran for 5 min after the addition of the enzyme (time 0). Spectra are shown every 1 min. (Inset) Initial rates as a function the NADPH concentration. The continuous curve was adjusted to the Michaelis-Menten equation by nonlinear least squares with GNUPLOT. (B) pH dependence of the MICAL reaction. (C) Inhibition by EGCG. The reaction rate was measured as a function of the NADPH concentration for concentrations of 0.0, 30.0, and 60.0 μM.
Scheme 1.
Scheme 1.
Fig. 6.
Fig. 6.
Charge distribution in the MICALfd surface. Positive areas are shown in blue, negative areas are shown in red, and neutral areas are shown in gray. The residues surrounding a strong positive charge feature assumed to be involved in NADPH binding are indicated. The yellow arrow indicates to the location of the FAD isoalloxazine ring.
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