Structural and Evolutionary Adaptation of NOD-Like Receptors in Birds

Abstract

NOD-like receptors (NLRs) are intracellular sensors of the innate immune system that recognize intracellular pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs). Little information exists regarding the incidence of positive selection in the evolution of NLRs of birds or the structural differences between bird and mammal NLRs. Evidence of positive selection was identified in four avian NLRs (NOD1, NLRC3, NLRC5, and NLRP3) using the maximum likelihood approach. These NLRs are under different selection pressures which is indicative of different evolution patterns. Analysis of these NLRs showed a lower percentage of codons under positive selection in the LRR domain than seen in the studies of Toll-like receptors (TLRs), suggesting that the LRR domain evolves differently between NLRs and TLRs. Modeling of human, chicken, mammalian, and avian ancestral NLRs revealed the existence of variable evolution patterns in protein structure that may be adaptively driven.

Figure 1

Adaptive substitutions in avian NLRs NACHT domain. We have aligned the NACHT domain of NLRs (human and Gallus shown). Adaptive amino acid substitutions and important motif of NACHT are indicated along the top of the alignment. Adaptive amino acid substitutions in NACHT are represented with star (^∗); motifs involved in ATP binding and hydrolysis of NACHT are indicated along the top of the alignment.

Figure 2

Sequence and primary functional differences in NLRC5 CARD are established (human, chicken, mouse, mammalian ancestor, and avian ancestor). (a) Sequence alignments of NLRC5 CARD domain. Adaptive amino acid substitutions in birds are represented with star (^∗). Secondary structures of protein are shown along the top of the alignment. NLS (amino acids 121 to 134 in human) are shown. (b–e) The atypical CARD of NLRC5 consists of five α-helices (α1, α2, α4, α5, and α6) that are packed around a hydrophobic core. The CARD shape and electrostatic potential for human, chicken, mouse, mammalian ancestor, and avian ancestor are plotted. The surfaces are color-coded according to electrostatic surface potential: red, −10 kT; white, 0 kT; and blue, +10 kT. (f) Structural alignment of human, chicken, mammalian ancestor, and avian ancestor NLRC5 atypical CARD.

Figure 3

Location of positively selected sites of four NLR molecules in birds. Protein architectures were generated with PROSITE MyDomains image creator tool (http://www.expasy.org/tools/mydomains/). Red bars indicate adaptive amino acid substitutions. Colored areas represent different conserved domains (blue, CARD or PYD domain; green, NACHT domain; orange, LRR domain).

Figure 4

Sequence and primary functional differences in NOD1 CARD. (a) Alignment of NOD1 CARD domain sequences. Star (^∗) indicates adaptive amino acid substitutions of NOD1 CARD in birds. (b) The NOD1 CARD shape and electrostatic potential for human, chicken, mouse, mammalian ancestor, and avian ancestor are plotted; the surfaces are color-coded according to electrostatic surface potential: red, −10 kT; white, 0 kT; and blue, +10 kT. Residues reported to be important for NOD1/RIP2 interaction, and NF-κB activation in human is labeled.

Figure 5

WebLogo of the putative TRAF2-binding sites constructed from alignment of 41 birds NLRC3 sequences. The residue numbering corresponds to the residues from chicken NLRC3. TRAF-binding motif mainly contains four residues with (P/S/A/T)-X-(Q/E)-E in the NACHT domain; “P/S/A/T” represents Pro, Ser, Ala, or Thr; “X” indicates any amino acid; “Q/E” represents Gln or Glu. Major TRAF2-binding motifs in birds NLRC3 are at the region of 483-486 and 608-611. The letter size is proportional to the degree of amino acid conservation. The weblogo was made using the web-based application WebLogo (http://weblogo.berkeley.edu).