TRIM21 is an IgG receptor that is structurally, thermodynamically, and kinetically conserved - PubMed (original) (raw)

Comparative Study

. 2008 Apr 22;105(16):6045-50.

doi: 10.1073/pnas.0800159105. Epub 2008 Apr 17.

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Comparative Study

TRIM21 is an IgG receptor that is structurally, thermodynamically, and kinetically conserved

Anthony H Keeble et al. Proc Natl Acad Sci U S A. 2008.

Abstract

The newly identified tripartite motif (TRIM) family of proteins mediate innate immunity and other critical cellular functions. Here we show that TRIM21, which mediates the autoimmune diseases rheumatoid arthritis, systemic lupus erythematosus, and Sjögren's syndrome, is a previously undescribed IgG receptor with a binding mechanism unlike known mammalian Fcgamma receptors. TRIM21 simultaneously targets conserved hot-spot residues on both Ig domains of the Fc fragment using a PRYSPRY domain with a preformed multisite interface. The binding sites on both TRIM21 and Fc are highly conserved to the extent that the proteins are functionally interchangeable through murine, canine, primate, and human species. Pre-steady-state analysis exposes mechanistic conservation at the level of individual residues, which make the same energetic and kinetic contributions to binding despite varying in sequence. Together, our results reveal that TRIM21 is a previously undescribed type of IgG receptor based on a non-Ig scaffold whose interaction at the fundamental level-structural, thermodynamic, and kinetic-is evolutionarily conserved.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Comparison of free and bound mouse TRIM21 structures. (Left) Superposition of free (wheat) with bound (orange) PRYSPRY domain. Binding-site loops and residues making important contributions are annotated. (Right) Superposition of TRIM21:Fc complex (orange and red, respectively) with one Fc heavy chain from mouse IgG structure 1IGT (24) (pink). A superposition of the free structure is shown in yellow.

Fig. 2.

Fig. 2.

Pre-steady-state kinetics of mouse TRIM21:Fc interaction. (A) Stopped-flow spectrofluorimetry was used to measure the rapid fluorescence quench upon mouse TRIM21–mouse IgG association (see Materials and Methods for details). The data (red circles) fit to a single exponential (solid black line). Linear regression of the linear increase in _k_obs (Inset) yields an association rate constant (_k_on) of 3.16 × 106 M−1s−1. (B) Measurement of the dissociation rate constant (_k_off) for mouse TRIM21–mouse IgG binding in a stopped-flow experiment by competition with excess protein A. This yields a _k_off of 1.35 s−1 resulting in a kinetic _K_d of 437 nM, close to that observed by ITC.

Fig. 3.

Fig. 3.

Hot-spot, electrostatic potential, and hydrophobicity conservation in the TRIM21 PRYSPRY binding site. Human (Left) is compared with mouse (Right). The molecular surface of the binding site is shown and colored on a red scale for ΔΔG, on a green scale for hydrophobicity, and on a red to blue (negative to positive) scale for electrostatic potential.

Fig. 4.

Fig. 4.

Effect of site-directed mutagenesis on cognate versus noncognate species TRIM21:IgG interaction. When the effects of mouse TRIM21 mutations on binding mouse IgG (cognate; x axis) are plotted against the effect on binding human IgG (noncognate; y axis) there exists a striking linear correlation (_R_2 = 0.98) suggesting that the same interactions are used to stabilize both complexes.

Fig. 5.

Fig. 5.

Sequence alignment of mouse and human TRIM21. Identical residues are shaded gray. The positions of the variable binding loops are shaded purple.

Fig. 6.

Fig. 6.

Comparison of mouse and human TRIM21. (Left) Superposition of mouse (orange) and human (yellow) PRYSPRY domains. Where the sequences diverge the annotations are formatted so that the first residue is for human and the second is for mouse; e.g., D/S326 indicates an aspartic acid in human and a serine in mouse. (Right) Superposition of mouse (red and orange) and human (pink and yellow) complexes with Fc.

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