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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Immunity. Author manuscript; available in PMC 2013 May 20.
Published in final edited form as:
PMCID: PMC3658283
NIHMSID: NIHMS363731

NLRX1 has a Tail to Tell

Abstract

In this issue of Immunity, Hong et al. (2012) report the first structural analysis of the C-terminal fragment of an NLR [nucleotide-binding domain (NBD) andleucine-rich repeat (LRR) containing] protein, NLRX1. This fragment forms a hexamer and binds RNA.

The NLRs (nucleotide-binding domain (NBD) and leucine-rich repeat (LRR) containing proteins) are a large protein family that plays important roles in a wide-range of biological processes,a prime function of whichis the detection of pathogen associated molecular patterns (PAMPs). NLRX1 is the first NLR protein shown to be localized at the mitochondria. It has been associated with multiple functions, including negatively impacting anti-viral inflammatory response via the MAVS-RIG-Isensing pathway(Moore et al., 2008; Xia et al., 2011), or lipopolisaccharide-elicited response via the TRAF6-IKK signaling pathway(Allen et al., 2011; Xia et al., 2011), and positively controlling NF-κB and JNKsignaling pathwayto activatereactive oxygen species (ROS) production in response to TNF-α, poly(I:C) and pathogens(Abdul-Sater et al., 2010; Tattoli et al., 2008). Gene deletion of exons 4 and 5 within the Nlrx1gene resulted in elevated type I interferonand IL-6 response to viral infection incells and whole animals, and a modest reduction of basal ROS in innate immune cells(Allen et al., 2011). A different gene targeted strain showed reduced ROS production to Chlamydia, although the deleted genetic sequence was not described(Abdul-Sater et al., 2010).A third group studied a strain that has a deletion of the first four exons of Nlrx1(Rebsamen et al., 2011). These authors did not detect an effect of this deletion on either IFN expression or TNF-α amplification of NF-κB and JNK signaling. In addition to possibly mediating different functions, the precise site of cellular localization varies in different reports. Althoughone group has found endogenous NLRX1 localization as a mitochondrial outer membrane (MOM) protein(Moore et al., 2008), another group has shown its localization in the mitochondrial matrix(Arnoult et al., 2009). Different serologic reagentswere used in these localization studies, which might impact the outcome. Additionally,one has to entertain that these varied localizationsmay be relevant to the multiple functions NLRX1 reported to date.

Hong et al. (2012) in this issue of Immunitypresents the first crystallographic analysis of the C-terminal fragment of NLRX1 that includes two α helical domains flanking a middle LRR section. Several important and surprising features of the NLRX1 are revealed in this study. A key finding is that the NLRX1 LRR and its associated helical domains form a hexameric platform composed of a trimer of three dimers, through extensive intramolecular and intermolecular interactions (Figure 1, insert). These higher order oligomers may promote cooperative association between NLRX1 and its target proteins, thus ensure stringent regulation of innate immune responses. Althoughthe NLR nucleotide-binding domain (NBD) is typically considered an oligomerization motif, an early report that first described oligomerization of the founding NLR protein, CIITA, revealed that both NBD and LRR domains undergo homotypic (NBD-NBD, LRR-LRR) and heterotypic association (NBD-LRR) (Linhoff et al., 2001). In agreement with this, Hong et al. shows that LRR-LRR interaction occurs through theC-terminal α-helical domain (referred to as LRRCT by these authors) mediated intra- and intermolecular interactions at the dimer interface, and the N-terminal α-helical domain (referred to as LRRNT) mediated intermolecular interactions at the trimer interface. Whether the NLRX1 NBD domain promotes further oligomerization of NLRX1 is unknown. Interestingly, the NLRX1 NBD has a classic Walker A motif that binds ATPbut a poor Walker B motif. It is possible that an uncommon ATP binding motif endows different oligomerization function for the NLRX1 NBD.

Figure 1
A Working Model of NLRX1 Function

A second finding in this paper relates to how NLRs detect PAMPs, a central unresolved issue in the field. One of the most-studied NLRs is NLRP3, which mediates inflammasomeresponse to numerous PAMPs and DAMPs (damage-associated molecular patterns). An emerging consensus is that NLRP3 is unlikely to directly bind to such a wide variety of molecular structures. Rather it might“sense”through indirect detectionof PAMPs and DAMPs by an intermediate protein(s) that remains elusive. A recent large-scale analysis of plant NLRs has been illuminating regarding the intersection of NLRs and PAMPs. This work shows that only 2 of 30 NLRs directly interact with PAMPs (pathogen effectors)(Mukhtar et al., 2011). By contrast, nearly half of the host cell proteins that interact with NLRs are direct targets of pathogen effectors. These findings led to the conclusion that a majorityof pathogen effectors interact with host proteins that in turn activate plant NLRs, while a minority directly associate with NLRs. Support for the latter is emerging in mammalian cellsalthough direct binding between NLRs and their respective ligands have yet to be demonstrated in a purified recombinant system.

Hong et al., used a purified recombinant C-terminal fragment of NLRX1 to demonstrate its binding to ssRNA, dsRNA and poly(I:C), but not DNA. Based on the X-ray crystallographic analysis, they identified a large continuous positively-charged surface patch that might represent the RNA binding face. A single-residue mutation within this patch greatly reduced RNA binding by purified NLRX1 in a gel-shift assay. This mutant protein failed to induce ROS upon stimulation with poly(I:C).However, further characterization of NLRX1 co-localization with RNA within cells are important for physiological verification, and co-crystallization of NLRX1 with RNA would be a strong confirmation of this interaction. Finally exactly where NLRX1 binds RNA within the cell is important. As mentioned earlier, NLRX1 has been localized to the MOM and mitochondria matrix in separate studies. Specifically, NLRX1 interacts with a mitochondrial matrix protein, UQCRC2, which is a component of the respiratory chain complex III, thus providing a possible basis for ROS induction by NLRX1 (Arnoult et al., 2009; Rebsamen et al., 2011) (Figure 1). The current work used ROS induction upon poly(I:C) stimulation as a readout. While it is conceivable that viral RNA bind to NLRX1 at the MOM, it is more difficult to envision how NLRX1 can access viral RNA from the mitochondria matrix. However as revealed by this work, there is much that we do not understand about this fascinating protein, and only additional studies can clarify these issues.

Exactly how RNA binding elicits the biological function of NLRX1 is unclear.Does RNA-binding to NLRX1 influence NLRX1-UQCRC2 association to affect ROS production? One report showed that endogenous NLRX1 constitutively interacts with MAVS(Moore et al., 2008)Does RNA induce conformational changes or oligomerization states of NLRX1 so that NLRX1 releases MAVS to allow for RIG-I signaling? The hexameric full-length NLRX1 model shows close proximity of its RNA binding surface with the modeled NBD domains. It is conceivable that RNA-binding at LRR and MAVS binding at NBD are mutually exclusive, thus providing a mechanism to release the repression of the RIG-I pathway upon RNA exposure to both RIG-I and NLRX1. The above working models can now be tested with the information provided by Hong et al.

Footnotes

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