Structural Determinants at the Interface of the ARC2 and Leucine-Rich Repeat Domains Control the Activation of the Plant Immune Receptors Rx1 and Gpa2

Many plant and animal immune receptors have a modular nucleotide-binding-leucine-rich repeat (NB-LRR) architecture in which a nucleotide-binding switch domain, NB-ARC, is tethered to a LRR sensor domain. The cooperation between the switch and sensor domains, which regulates the activation of these proteins, is poorly understood. Here, we report structural determinants governing the interaction between the NB-ARC and LRR in the highly homologous plant immune receptors Gpa2 and Rx1, which recognize the potato cyst nematode Globodera pallida and Potato virus X, respectively. Systematic shuffling of polymorphic sites between Gpa2 and Rx1 showed that a minimal region in the ARC2 and N-terminal repeats of the LRR domain coordinate the activation state of the protein. We identified two closely spaced amino acid residues in this region of the ARC2 (positions 401 and 403) that distinguish between autoactivation and effector-triggered activation. Furthermore, a highly acidic loop region in the ARC2 domain and basic patches in the N-terminal end of the LRR domain were demonstrated to be required for the physical interaction between the ARC2 and LRR. The NB-ARC and LRR domains dissociate upon effector-dependent activation, and the complementary-charged regions are predicted to mediate a fast reassociation, enabling multiple rounds of activation. Finally, we present a mechanistic model showing how the ARC2, NB, and N-terminal half of the LRR form a clamp, which regulates the dissociation and reassociation of the switch and sensor domains in NB-LRR proteins.Resistance (R) proteins play a central role in the recognition-based immune system of plants. Unlike vertebrates, plants lack an adaptive immune system with highly specialized immune cells. Instead, they rely on an innate immune system in which each cell is autonomous. Two types of immune receptors can be distinguished in plants, pathogen-associated molecular patterns recognition receptors that detect conserved molecular patterns in plant pathogens and intracellular R proteins that recognize specific effectors employed by pathogens as modifiers of host metabolism or defense mechanisms (Jones and Dangl, 2006). Effector-triggered activation of R proteins leads to an array of protective responses, often culminating in programmed cell death at the site of infection (Greenberg and Yao, 2004), thereby preventing further ingress of the pathogen. Pathogens have evolved mechanisms to evade recognition by R proteins and to regain their virulence (Dodds and Rathjen, 2010). This continuous coevolutionary process between host and pathogen has resulted in a reservoir of highly diverse R proteins in plants, enabling them to counteract a wide range of pathogens and pests.The most common class of R proteins consists of nucleotide-binding (NB)-leucine-rich repeat (LRR) proteins with a tripartite domain architecture, which roughly corresponds to an N-terminal response domain (a coiled coil [CC] or Toll/Interleukin-1 receptor [TIR] domain) involved in downstream signaling, a central molecular switch domain (the NB domain present in the mammalian apoptosis regulator Apaf1, plant R proteins, and the Caenorhabditis elegans apoptosis regulator CED4 [NB-ARC]), and a C-terminal sensor domain (the LRR domain). The NB-ARC domain is an extended nucleotide-binding domain that plant immune receptors share with metazoan apoptosis regulators and immune receptors such as Apaf1, CED4, and nucleotide-binding oligomerization domain (NOD-like) receptors (NLRs) and belongs to the STAND (signal transduction ATPases with numerous domains) family of nucleoside triphosphatase domains (van der Biezen and Jones, 1998; Leipe et al., 2004; Albrecht and Takken, 2006; Maekawa et al., 2011b). The overall modular architecture of metazoan STAND nucleoside triphosphatase is similar to that of NB-LRR plant immune receptors, but the domains flanking the NB-ARC domain often differ. In NLRs, for example, several N-terminal domains can be found, including caspase-recruiting domains and Pyrin domains (Proell et al., 2008). In the mammalian protein Apaf1, the sensor involved in cytochrome c detection consists of C-terminal WD40 repeats (Zou et al., 1997).In plant NB-LRR resistance proteins, the recognition of a pathogen effector via the LRR domain is thought to switch the conformation of the protein from a closed, autoinhibited “off” state into an open, active “on” state (Lukasik and Takken, 2009). The activation of NB-LRR proteins is most likely a multistep process in which the NB-ARC domain plays a central role. The three subdomains of the NB-ARC, the NB, ARC1, and ARC2, collectively form a nucleotide-binding pocket that adopts different conformations depending on the bound nucleotide. This mechanism seems to be conserved between proteins from organisms as distant as bacteria, metazoans, and plants (Rairdan and Moffett, 2007; Danot et al., 2009; Takken and Tameling, 2009). The conformational change coincides with the exchange of bound ADP for ATP in the NB-ARC, probably stabilizing the active conformation (Tameling et al., 2006; Ade et al., 2007). Hydrolysis of the bound ATP is hypothesized to return the domains to their inactive state. The exact mechanism by which elicitor recognition via the LRR leads to a conformational change of the NB-ARC and the subsequent activation of immune signaling pathways is not clear.Previous studies have shown that the CC/TIR, NB-ARC, and LRR domains in plant immune receptors interact and cooperate with each other in an interdependent manner (Moffett et al., 2002; Leister et al., 2005; Ade et al., 2007; Rairdan et al., 2008). From these data, a picture emerges in which the LRR domain is not only involved in pathogen recognition, but also plays a role in maintaining an autoinhibited resting state in the absence of pathogens via its interactions with the other domains (Bendahmane et al., 2002; Hwang and Williamson, 2003; Ade et al., 2007; Qi et al., 2012). A similar role as regulatory domain has been found for the sensor domains of other NLRs, such as the mammalian Apaf1 (Hu et al., 1998). For the potato (Solanum tuberosum) immune receptor Rx1, a model plant NB-LRR protein, it has been shown that the LRR cooperates with the ARC subdomains in retaining the inactive state of the protein. The deletion of the ARC and LRR domains leads to a constitutive activity of the NB (Bendahmane et al., 2002; Rairdan et al., 2008). In addition, it was demonstrated that the elicitor, the Potato virus X (PVX) coat protein, modifies the interdomain interactions in Rx1 (Moffett et al., 2002; Rairdan et al., 2008). Sequence exchanges between Rx1 and the highly homologous nematode resistance protein Gpa2 (88% amino acid identity) resulted in incompatibilities between the domains that give rise to inappropriate activation of cell death responses (Rairdan and Moffett, 2006), indicating that the cooperation between the sensor and switch domains depends on an interaction fine tuned by intramolecular coevolution. In this light, it is interesting to note that a functional ortholog of Rx1, Rx2 from Solanum acaule, is almost identical to Rx1 in its LRR region but displays a higher similarity to Gpa2 in stretches of its CC-NB-ARC sequence (Bendahmane et al., 2000).The aim of our study was to pinpoint the molecular determinants controlling the switch between the resting and activation state of NB-LRR proteins. The incompatibility between the ARC and LRR domains of Rx1 and Gpa2 was used as a guideline to dissect the molecular and structural determinants involved in the cooperation between the switch (NB-ARC) and sensor (LRR) domain. An extensive exchange of polymorphic residues between these two homologous NB-LRR proteins resulted in the identification of a minimal fragment of 68 amino acid residues in the ARC2 domain and the first LRR repeats as being crucial for proper activation of Gpa2 and Rx1. Within this minimal region, we identified two amino acids that, despite their proximity in the amino acid sequence, differentiate between elicitor-dependent (position 401) and independent activation (position 403). However, structural modeling of the domains shows that the residue at position 403 operates at the interface of the ARC2 and N-terminal part of the LRR domain, while residue 401 mapped at the interface between the ARC2 and NB domain. Furthermore, an acidic loop region in the ARC2 domain and complementary-charged basic patches in the N-terminal half of the LRR domain are shown to be required for the physical interaction between these domains. We demonstrate that the binding between the CC- NB-ARC and LRR domains is disrupted upon elicitor-dependent activation and that the complementary-charged residues are predicted to facilitate reassociation. Two independent docking simulations of the NB-ARC and LRR domain indicate that the LRR domain binds to the NB-ARC domain at the surface formed by the interaction of the ARC2 and NB subdomains. We present a mechanistic model in which the first repeats of the LRR, the ARC2 subdomain, and the NB form a clamp, which governs the shuttling between a closed, autoinhibited “off” state and an open, active “on” state of the resistance protein. Finally, we discuss the consequences of the functional constraints imposed by the interface of the NB, ARC2, and LRR domain for the generation of novel resistance specificities via evolutionary processes and genetic engineering.     

Computational biomechanics of a lumbar motion segment in pure and combined shear loads

Anterior shear has been implicated as a risk factor in spinal injuries. A 3D nonlinear poroelastic finite element model study of a lumbar motion segment L4-L5 was performed to predict the temporal shear response under various single and combined shear loads. Effects of nucleotomy and facetectomy as well as changes in the posture and facet gap distance were analyzed as well.     

Ascorbyl palmitate interaction with phospholipid monolayers: Electrostatic and rheological preponderancy

Ascorbyl palmitate (ASC₁₆) is an anionic amphiphilic molecule of pharmacological interest due to its antioxidant properties. We found that ASC₁₆ strongly interacted with model membranes. ASC₁₆ penetrated phospholipid monolayers, with a cutoff near the theoretical surface pressure limit. The presence of a lipid film at the interface favored ASC₁₆ insertion compared with a bare air/water surface. The adsorption and penetration time curves showed a biphasic behavior: the first rapid peak evidenced a fast adsorption of charged ASC₁₆ molecules to the interface that promoted a lowering of surface pH, thus partially neutralizing and compacting the film. The second rise represented an approach to the equilibrium between the ASC₁₆ molecules in the subphase and the surface monolayer, whose kinetics depended on the ionization state of the film. Based on the Langmuir dimiristoylphosphatidylcholine + ASC₁₆ monolayer data, we estimated an ASC₁₆ partition coefficient to dimiristoylphosphatidylcholine monolayers of 1.5 × 10⁵ and a ΔG_p = − 6.7 kcal·mol^− 1. The rheological properties of the host membrane were determinant for ASC₁₆ penetration kinetics: a fluid membrane, as provided by cholesterol, disrupted the liquid-condensed ASC₁₆-enriched domains and favored ASC₁₆ penetration. Subphase pH conditions affected ASC₁₆ aggregation in bulk: the smaller structures at acidic pHs showed a faster equilibrium with the surface film than large lamellar ones. Our results revealed that the ASC₁₆ interaction with model membranes has a highly complex regulation. The polymorphism in the ASC₁₆ bulk aggregation added complexity to the equilibrium between the surface and subphase form of ASC₁₆, whose understanding may shed light on the pharmacological function of this drug.     

Alexander Böhm (1971–2012)

On November 28, 2012 Alexander (Alex) Böhm, a bacterial geneticist, died at age 41, only a few months after taking up a position as an assistant professor at the LOEWE Center for Synthetic Microbiology in Marburg, Germany. Earlier in 2012 Alex had been diagnosed with an aggressive form of thyroid cancer that left him little time to live his scientific and personal dreams.     

Pediatric Invasive Pneumococcal Disease Caused by Vaccine Serotypes following the Introduction of Conjugate Vaccination in Denmark

A seven-valent pneumococcal conjugate vaccine (PCV7) was introduced in the Danish childhood immunization program (2+1 schedule) in October 2007, followed by PCV13 starting from April 2010. The nationwide incidence of IPD among children younger than 5 years nearly halved after the introduction of PCV7 in the program, mainly due to a decline in IPD caused by PCV7-serotypes. We report the results from a nationwide population-based cohort study of laboratory confirmed IPD cases in children younger than 5 years during October 1, 2007 to December 31, 2010 and describe the characteristics of children suspected to present with a vaccine failure. The period between April 19 and December 31, 2010 was considered a PCV7/PCV13 transitional period, where both vaccines were offered. We identified 45 episodes of IPD caused by a PCV7 serotype (23% of the total number) and 105 (55%) caused by one of the 6 additional serotypes in PCV13. Ten children had received at least one PCV7 dose before the onset of IPD caused by a PCV7 serotype. Seven children were considered to be incompletely vaccinated before IPD, but only three cases fulfilled the criteria of vaccine failure (caused by serotypes 14, 19F and 23F). One case of vaccine failure was observed in a severely immunosuppressed child following three PCV7 doses, and two cases were observed in immunocompetent children following two infant doses before they were eligible for their booster. None of the IPD cases caused by the additional PCV13 serotypes had been vaccinated by PCV13 and there were therefore no PCV13-vaccine failures in the first 8-months after PCV13 introduction in Denmark.     

Effects of population size and mutation rate on the evolution of mutational robustness

It is often assumed that the efficiency of selection for mutational robustness would be proportional to mutation rate and population size, thus being inefficient in small populations. However, Krakauer and Plotkin (2002) hypothesized that selection in small populations would favor robustness mechanisms, such as redundancy, that mask the effect of deleterious mutations. In large populations, by contrast, selection is more effective at removing deleterious mutants and fitness would be improved by eliminating mechanisms that mask the effect of deleterious mutations and thus impede their removal. Here, we test whether these predictions are supported in experiments with evolving populations of digital organisms. Digital organisms are self-replicating programs that inhabit a virtual world inside a computer. Like their organic counterparts, digital organisms mutate, compete, evolve, and adapt by natural selection to their environment. In this study, 160 populations evolved at different combinations of mutation rate and population size. After 10(4) generations, we measured the mutational robustness of the most abundant genotype in each population. Mutational robustness tended to increase with mutation rate and to decline with population size, although the dependence with population size was in part mediated by a negative relationship between fitness and robustness. These results are independent of whether genomes were constrained to their original length or allowed to change in size.     

Thermodynamics of neutral protein evolution

Naturally evolving proteins gradually accumulate mutations while continuing to fold to stable structures. This process of neutral evolution is an important mode of genetic change and forms the basis for the molecular clock. We present a mathematical theory that predicts the number of accumulated mutations, the index of dispersion, and the distribution of stabilities in an evolving protein population from knowledge of the stability effects (delta deltaG values) for single mutations. Our theory quantitatively describes how neutral evolution leads to marginally stable proteins and provides formulas for calculating how fluctuations in stability can overdisperse the molecular clock. It also shows that the structural influences on the rate of sequence evolution observed in earlier simulations can be calculated using just the single-mutation delta deltaG values. We consider both the case when the product of the population size and mutation rate is small and the case when this product is large, and show that in the latter case the proteins evolve excess mutational robustness that is manifested by extra stability and an increase in the rate of sequence evolution. All our theoretical predictions are confirmed by simulations with lattice proteins. Our work provides a mathematical foundation for understanding how protein biophysics shapes the process of evolution.     

Adipose tissue interleukin-18 mRNA and plasma interleukin-18: effect of obesity and exercise

Objectives: Obesity and a physically inactive lifestyle are associated with increased risk of developing insulin resistance. The hypothesis that obesity is associated with increased adipose tissue (AT) interleukin (IL)‐18 mRNA expression and that AT IL‐18 mRNA expression is related to insulin resistance was tested. Furthermore, we speculated that acute exercise and exercise training would regulate AT IL‐18 mRNA expression. Research Methods and Procedures: Non‐obese subjects with BMI < 30 kg/m² (women: n = 18; men; n = 11) and obese subjects with BMI >30 kg/m² (women: n = 6; men: n = 7) participated in the study. Blood samples and abdominal subcutaneous AT biopsies were obtained at rest, immediately after an acute exercise bout, and at 2 hours or 10 hours of recovery. After 8 weeks of exercise training of the obese group, sampling was repeated 48 hours after the last training session. Results: AT IL‐18 mRNA content and plasma IL‐18 concentration were higher (p < 0.05) in the obese group than in the non‐obese group. AT IL‐18 mRNA content and plasma IL‐18 concentration was positively correlated (p < 0.05) with insulin resistance. While acute exercise did not affect IL‐18 mRNA expression at the studied time‐points, exercise training reduced AT IL‐18 mRNA content by 20% in both sexes. Discussion: Because obesity and insulin resistance were associated with elevated AT IL‐18 mRNA and plasma IL‐18 levels, the training‐induced lowering of AT IL‐18 mRNA content may contribute to the beneficial effects of regular physical activity with improved insulin sensitivity.