Single Molecule Force Spectroscopy of the Cardiac Titin N2B Element: EFFECTS OF THE MOLECULAR CHAPERONE ��B-CRYSTALLIN WITH DISEASE-CAUSING MUTATIONS*

The small heat shock protein αB-crystallin interacts with N2B-Us, a large unique sequence found in the N2B element of cardiac titin. Using single molecule force spectroscopy, we studied the effect of αB-crystallin on the N2B-Us and its flanking Ig-like domains. Ig domains from the proximal tandem Ig segment of titin were also studied. The effect of wild type αB-crystallin on the single molecule force-extension curve was determined as well as that of mutant αB-crystallins harboring the dilated cardiomyopathy missense mutation, R157H, or the desmin-related myopathy mutation, R120G. Results revealed that wild type αB-crystallin decreased the persistence length of the N2B-Us (from ∼0.7 to ∼0.2 nm) but did not alter its contour length. αB-crystallin also increased the unfolding force of the Ig domains that flank the N2B-Us (by 51 ± 3 piconewtons); the rate constant of unfolding at zero force was estimated to be ∼17-fold lower in the presence of αB-crystallin (1.4 × 10^-4 s^-1 versus 2.4 × 10^-3 s^-1). We also found that αB-crystallin increased the unfolding force of Ig domains from the proximal tandem Ig segment by 28 ± 6 piconewtons. The effects of αB-crystallin were attenuated by the R157H mutation (but were still significant) and were absent when using the R120G mutant. We conclude that αB-crystallin protects titin from damage by lowering the persistence length of the N2B-Us and reducing the Ig domain unfolding probability. Our finding that this effect is either attenuated (R157H) or lost (R120G) in disease causing αB-crystallin mutations suggests that the interaction between αB-crystallin and titin is important for normal heart function.αB-crystallin is a member of the small heat shock protein family that by inhibiting denaturation and aggregation of proteins functions as a molecular chaperone (1). Although αB-crystallin has been most intensively studied in the vertebrate eye lens, it is also found in many other tissues (2) with cardiac muscle expressing αB-crystallin at 3-5% of the total soluble protein (3). Up-regulation of αB-crystallin occurs in a number of cardiac disorders, including familial cardiac hypertrophy, and overexpression appears to protect the cardiac cell from ischemia reperfusion injury (for a review see Ref. 4). An important binding partner of αB-crystallin in cardiac muscle is titin (5, 6). Titin is a large filamentous protein that forms a continuous filament along the myofibril, with single titin molecules spanning from the edge to the middle of the sarcomere, a distance of ∼1 μm (7). The I-band region of titin is extensible and functions as a molecular spring that, when extended, develops force (8, 9). This force is an important determinant of the passive stiffness of the heart that determines the filling characteristics during the diastolic part of the heart cycle (10). The interaction between αB-crystallin and titin could be important for maintaining heart function, especially when stressed, such as during ischemia (5), warranting studies of the effect of αB-crystallin on the biomechanical properties of titin.The molecular spring region of titin contains three distinct spring elements (7). The first element is the tandem Ig segment, consisting of serially linked Ig domains that form the so-called proximal tandem Ig segment (15 Ig domains) near the Z-disk of the sarcomere and a distal segment (22 Ig domains) near the A-band (11). The second spring element is the PEVK, a unique sequence that contains largely prolines, glutamates, valines, and lysines (11). The third element consists of a large unique sequence (in human 572 residues in size) named the N2B-Us; it is heart-specific and dominates the extension of titin near the upper limit of the physiological sarcomere length range (12). αB-crystallin appears to preferentially bind to the N2B-Us, although weak binding to Ig domains has also been detected (6). Previous studies have shown that αB-crystallin increases the unfolding force of Ig 91-98, a fragment that contains eight Ig domains from the distal tandem Ig segment of titin (6). However, the mechanical effect of αB-crystallin on the N2B-Us (its main binding partner in titin) has not been investigated.The association between αB-crystallin and titin has prompted a search for disease causing mutations in αB-crystallin. This revealed in patients with dilated cardiomyopathy (DCM),² a missense mutation, R157H, that affects an evolutionarily conserved amino acid residue; the mutation decreases the binding to the N2B domain without affecting distribution of the mutant crystallin protein in cardiomyocytes (13). In another disease, the desmin-related myopathy mutation R120G (14) decreases the binding of αB-crystallin to the N2B element and causes intracellular aggregates of the mutant protein (13).In the present study, we used single molecule force spectroscopy and determined the contour length (CL; end-to-end length when stretched with infinite force) and persistence length (PL; a measure of the bending rigidity) of the N2B-Us. We also studied the unfolding force of Ig domains, those that flank the N2B-Us and those that make up the proximal tandem Ig segment. In addition, we investigated the effect of wild type and R157H and R120G αB-crystallin on the molecular mechanics of the N2B-Us, its flanking Ig domains, and the Ig domains in the proximal tandem Ig segment. Findings support that αB-crystallin functions as a chaperone that lowers the probability of Ig domain unfolding and the persistence length of the titin N2B-Us spring region. Importantly, this chaperone function is significantly reduced by the R157H mutation and abolished by the R120G mutation.     

Effect of Copper and Zinc on the Single Molecule Self-Affinity of Alzheimer’s Amyloid-β Peptides

The presence of trace concentrations of metallic ions, such as copper and zinc, has previously been shown to drastically increase the aggregation rate and neurotoxicity of amyloid-β (Aβ), the peptide implicated in Alzheimer’s disease (AD). The mechanism of why copper and zinc accelerate Aβ aggregation is poorly understood. In this work, we use single molecule force spectroscopy (SMFS) to probe the kinetic and thermodynamic parameters (dissociation constant, K_d, kinetic dissociation rate, k_off, and free energy, ΔG) of the dissociation of an Aβ dimer, the amyloid species which initiates the amyloid cascade. Our results show that nanomolar concentrations of copper do not change the single molecule affinity of Aβ to another Aβ peptide in a statistically significant way, while nanomolar concentrations of zinc decrease the affinity of Aβ-Aβ by an order of magnitude. This suggests that the binding of zinc ion to Aβ may interfere with the binding of Aβ-Aβ, leading to a lower self-affinity.     

Modulation of Kinesin’s Load-Bearing Capacity by Force Geometry and the Microtubule Track

Kinesin motors and their associated microtubule tracks are essential for long-distance transport of cellular cargos. Intracellular activity and proper recruitment of kinesins is regulated by biochemical signaling, cargo adaptors, microtubule-associated proteins, and mechanical forces. In this study, we found that the effect of opposing forces on the kinesin-microtubule attachment duration depends strongly on experimental assay geometry. Using optical tweezers and the conventional single-bead assay, we show that detachment of kinesin from the microtubule is likely accelerated by forces vertical to the long axis of the microtubule due to contact of the single bead with the underlying microtubule. We used the three-bead assay to minimize the vertical force component and found that when the opposing forces are mainly parallel to the microtubule, the median value of attachment durations between kinesin and microtubules can be up to 10-fold longer than observed using the single-bead assay. Using the three-bead assay, we also found that not all microtubule protofilaments are equivalent interacting substrates for kinesin and that the median value of attachment durations of kinesin varies by more than 10-fold, depending on the relative angular position of the forces along the circumference of the microtubule. Thus, depending on the geometry of forces across the microtubule, kinesin can switch from a fast detaching motor (median attachment duration <0.2 s) to a persistent motor that sustains attachment (median attachment duration >3 s) at high forces (5 pN). Our data show that the load-bearing capacity of the kinesin motor is highly variable and can be dramatically affected by off-axis forces and forces across the microtubule lattice, which has implications for a range of cellular activities, including cell division and organelle transport.     

Synergistic Interactions between Alzheimer’s Aβ40 and Aβ42 on the Surface of Primary Neurons Revealed by Single Molecule Microscopy

Two amyloid-β peptides (Aβ40 and Aβ42) feature prominently in the extracellular brain deposits associated with Alzheimer’s disease. While Aβ40 is the prevalent form in the cerebrospinal fluid, the fraction of Aβ42 increases in the amyloid deposits over the course of disease development. The low in vivo concentration (pM-nM) and metastable nature of Aβ oligomers have made identification of their size, composition, cellular binding sites and mechanism of action challenging and elusive. Furthermore, recent studies have suggested that synergistic effects between Aβ40 and Aβ42 alter both the formation and stability of various peptide oligomers as well as their cytotoxicity. These studies often utilized Aβ oligomers that were prepared in solution and at μM peptide concentrations. The current work was performed using physiological Aβ concentrations and single-molecule microscopy to follow peptide binding and association on primary cultured neurons. When the cells were exposed to a 1:1 mixture of nM Aβ40:Aβ42, significantly larger membrane-bound oligomers developed compared to those formed from either peptide alone. Fluorescence resonance energy transfer experiments at the single molecule level reveal that these larger oligomers contained both Aβ40 and Aβ42, but that the growth of these oligomers was predominantly by addition of Aβ42. Both pure peptides form very few oligomers larger than dimers, but either membrane bound Aβ40/42 complex, or Aβ40, bind Aβ42 to form increasingly larger oligomers. These findings may explain how Aβ42-dominant oligomers, suspected of being more cytotoxic, develop on the neuronal membrane under physiological conditions.     

Bruton’s Tyrosine Kinase Mediates the Synergistic Signalling between TLR9 and the B Cell Receptor by Regulating Calcium and Calmodulin

B cells signal through both the B cell receptor (BCR) which binds antigens and Toll-like receptors (TLRs) including TLR9 which recognises CpG DNA. Activation of TLR9 synergises with BCR signalling when the BCR and TLR9 co-localise within an auto-phagosome-like compartment. Here we report that Bruton’s tyrosine kinase (BTK) is required for synergistic IL6 production and up-regulation of surface expression of MHC-class-II, CD69 and CD86 in primary murine and human B cells. We show that BTK is essential for co-localisation of the BCR and TLR9 within a potential auto-phagosome-like compartment in the Namalwa human B cell line. Downstream of BTK we find that calcium acting via calmodulin is required for this process. These data provide new insights into the role of BTK, an important target for autoimmune diseases, in B cell activation.     

Vitamin D and melatonin protect the cell’s viability and ameliorate the CCl4 induced cytotoxicity in HepG2 and Hep3B hepatoma cell lines

Carbon tetrachloride (CCl₄) is widely used to induce liver toxicity in in vitro/in vivo models. Lipid peroxidation (LPO) begins with toxicity and affects cell viability. Recently, the beneficial effects of melatonin and Vitamin D on cell proliferation in human normal and cancer cells were found. This study was planned to evaluate antioxidant and cytoprotective activity of melatonin and Vitamin D in CCl₄ induced cytotoxicity in HepG2 and Hep3B hepatoma cell lines. Based on the cytotoxicity assay, melatonin and Vitamin D were evaluated for cytotoprotective potential against CCl₄ induced toxicity in HepG2 and Hep3B liver cell lines by monitoring cell viability, LPO and glutathione (GSH) level. Different dosages of CCl₄ (0.1, 0.2, 0.3 and 0.4 % v/v) were applied to HepG2 and Hep3B cells in order to determine the most toxic dosage of it in a time dependent manner. The same experiments were repeated with exogenously applied melatonin (MEL) and Vitamin D to groups treated with/without CCL₄. Cell viability was determined with MTT measurements at the 2nd, 24th and 48th h. GSH content and Malondialdehyde levels were measured from the cell lysates. As a result, both melatonin and Vitamin D administration during CCl₄ exposure protected liver cells from CCl₄ induced cell damage. Increase in LPO and decrease in GSH were found in the CCl₄ groups of both cells. Contrary to these results administration of MEL and Vitamin D on cells exhibited results similar to the control groups. Therefore, melatonin and Vitamin D might be a promising therapeutic agent in several toxic hepatic diseases.     

Fast-to-Slow Transition of Skeletal Muscle Contractile Function and Corresponding Changes in Myosin Heavy and Light Chain Formation in the R6/2 Mouse Model of Huntington’s Disease

Huntington´s disease (HD) is a hereditary neurodegenerative disease resulting from an expanded polyglutamine sequence (poly-Q) in the protein huntingtin (HTT). Various studies report atrophy and metabolic pathology of skeletal muscle in HD and suggest as part of the process a fast-to-slow fiber type transition that may be caused by the pathological changes in central motor control or/and by mutant HTT in the muscle tissue itself. To investigate muscle pathology in HD, we used R6/2 mice, a common animal model for a rapidly progressing variant of the disease expressing exon 1 of the mutant human gene. We investigated alterations in the extensor digitorum longus (EDL), a typical fast-twitch muscle, and the soleus (SOL), a slow-twitch muscle. We focussed on mechanographic measurements of excised muscles using single and repetitive electrical stimulation and on the expression of the various myosin isoforms (heavy and light chains) using dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of whole muscle and single fiber preparations. In EDL of R6/2, the functional tests showed a left shift of the force-frequency relation and decrease in specific force. Moreover, the estimated relative contribution of the fastest myosin isoform MyHC IIb decreased, whereas the contribution of the slower MyHC IIx isoform increased. An additional change occurred in the alkali MyLC forms showing a decrease in 3f and an increase in 1f level. In SOL, a shift from fast MyHC IIa to the slow isoform I was detectable in male R6/2 mice only, and there was no evidence of isoform interconversion in the MyLC pattern. These alterations point to a partial remodeling of the contractile apparatus of R6/2 mice towards a slower contractile phenotype, predominantly in fast glycolytic fibers.     

Participation of Jasmonate Signaling Components in the Development of Arabidopsis thaliana’s Salt Resistance Induced by H2S and NO Donors

Russian Journal of Plant Physiology - The effect of donors of hydrogen sulfide (50 μM sodium hydrosulfide NaHS) and nitric oxide (500&nbsp;μM sodium nitroprusside, SNP) on the salt...     

Modification of mouse A2M B (620-792) and A2M N (168-230) by malondialdehyde and acetaldehyde attenuates the proteinase and TGF-β1 binding ability of A2MB

Acetaldehyde and malondialdehyde react covalently with cellular proteins forming protein-malondialdehyde-acetaldehyde adducts thus modulating their biochemical functions. Alpha-2 macroglobulin, an acute phase protein produced by liver binds to cytokines, growth factors and neutralizes proteinases. In this study we examined the formation of MAA adducts of N-terminal and bait region of mouse A2M and their effect on modulating its proteinase and TGF-β1 binding activities. Adduct formation abrogated the binding of bait region with TGF-β1, trypsin, and elastase. TGF-β1 induced NO production was also suppressed. Acetaldehyde and MDA adduction of A2M may have physiological consequences in alcoholic patients.     

Identification of Iron-Sulfur (Fe-S) Cluster and Zinc (Zn) Binding Sites Within Proteomes Predicted by DeepMind’s AlphaFold2 Program Dramatically Expands the Metalloproteome

DeepMind’s AlphaFold2 software has ushered in a revolution in high quality, 3D protein structure prediction. In very recent work by the DeepMind team, structure predictions have been made for entire proteomes of twenty-one organisms, with >360,000 structures made available for download. Here we show that thousands of novel binding sites for iron-sulfur (Fe-S) clusters and zinc (Zn) ions can be identified within these predicted structures by exhaustive enumeration of all potential ligand-binding orientations. We demonstrate that AlphaFold2 routinely makes highly specific predictions of ligand binding sites: for example, binding sites that are comprised exclusively of four cysteine sidechains fall into three clusters, representing binding sites for 4Fe-4S clusters, 2Fe-2S clusters, or individual Zn ions. We show further: (a) that the majority of known Fe-S cluster and Zn binding sites documented in UniProt are recovered by the AlphaFold2 structures, (b) that there are occasional disputes between AlphaFold2 and UniProt with AlphaFold2 predicting highly plausible alternative binding sites, (c) that the Fe-S cluster binding sites that we identify in E. coli agree well with previous bioinformatics predictions, (d) that cysteines predicted here to be part of ligand binding sites show little overlap with those shown via chemoproteomics techniques to be highly reactive, and (e) that AlphaFold2 occasionally appears to build erroneous disulfide bonds between cysteines that should instead coordinate a ligand. These results suggest that AlphaFold2 could be an important tool for the functional annotation of proteomes, and the methodology presented here is likely to be useful for predicting other ligand-binding sites.     

Independent ��-Arrestin2 and Gq/Protein Kinase C�� Pathways for ERK Stimulated by Angiotensin Type 1A Receptors in Vascular Smooth Muscle Cells Converge on Transactivation of the Epidermal Growth Factor Receptor

Recent studies in receptor-transfected cell lines have demonstrated that extracellular signal-regulated kinase (ERK) activation by angiotensin type 1A receptor and other G protein-coupled receptors can be mediated by both G protein-dependent and β-arrestin-dependent mechanisms. However, few studies have explored these mechanisms in primary cultured cells expressing endogenous levels of receptors. Accordingly, here we utilized the β-arrestin biased agonist for the angiotensin type 1A receptor, SII-angiotensin (SII), and RNA interference techniques to investigate angiotensin II (ANG)-activated β-arrestin-mediated mitogenic signaling pathways in rat vascular smooth muscle cells. Both ANG and SII induced DNA synthesis via the ERK activation cascade. Even though SII cannot induce calcium influx (G protein activation) after receptor stimulation, it does cause ERK activation, although less robustly than ANG. Activation by both ligands is diminished by depletion of β-arrestin2 by small interfering RNA, although the effect is more complete with SII. ERK activation at early time points but not later time points is strongly inhibited by those protein kinase C inhibitors that can block protein kinase Cζ. Moreover, ANG- and SII-mediated ERK activation require transactivation of the epidermal growth factor receptor via metalloprotease 2/9 and Src kinase. β-Arrestin2 facilitates ANG and SII stimulation of Src-mediated phosphorylation of Tyr-845 on the EGFR, a known site for Src phosphorylation. These studies delineate a convergent mechanism by which G protein-dependent and β-arrestin-dependent pathways can independently mediate ERK-dependent transactivation of the EGFR in vascular smooth muscle cells thus controlling cellular proliferative responses.G protein-coupled receptors, also known as seven transmembrane (7TM)² receptors, control virtually all known physiological processes in mammals (1). The various functions of these receptors are mediated and modulated by three families of proteins, which share the property that they interact virtually universally with the receptors in a strictly stimulus-dependent way (1). These three families of proteins are the heterotrimeric G proteins, the G protein-coupled receptor kinases (GRKs), and the β-arrestins. Activation of the receptors stimulates classical G protein-dependent signaling, often involving regulation of levels of second messengers such as cAMP and diacyglycerol. However, as has been known for many years, interaction of activated receptors with GRKs leading to their phosphorylation, and subsequent interaction with β-arrestins leads to desensitization of G protein signaling.In recent years, however, it has become increasingly clear that the β-arrestin-GRK system is in fact bifunctional (2). Thus, even as it desensitizes G protein signaling by the receptors, it also serves as a signal transduction system in its own right, activating a growing list of signaling pathways. These positive signaling functions are often mediated by the ability of β-arrestin to serve as an adaptor or scaffold molecule, bringing elements of diverse signaling pathways into proximity with one another and the receptors and thereby facilitating their activation. This new paradigm for understanding the previously unrecognized signaling properties of the β-arrestin-GRK system has been explored in a wide variety of transfected cultured cell systems.However, to date, relatively little investigation of these novel signaling pathways has been carried out in primary cell culture systems expressing endogenous levels of 7TM receptors. In seeking such a system in which to characterize and compare β-arrestin and G protein-mediated signaling pathways from a typical 7TM receptor, our attention was drawn to cultured rat vascular smooth muscle cells (VSMCs). Several features of rat VSMCs suggest this to be a relevant system for these purposes. Rat VSMCs express a variety of physiologically important 7TM receptors including the angiotensin II type 1A receptor (AT1R) (3). This receptor has been the focus of extensive study in transfected cell systems with respect to its β-arrestin-mediated signaling to a variety of pathways, most particularly extracellular signal-regulated kinase (ERK). Moreover, the AT1R mediates the physiologically important effects of angiotensin II (ANG) on vascular tone as well as on proliferation and chemotaxis (4, 5). Pathophysiologically, ANG stimulation of this receptor has been implicated in VSMC proliferation and chemotaxis, which are thought to play an important role in such important disease processes as atherosclerosis and restenosis after angioplasty (6, 7). Moreover, a ligand has been characterized [Sar¹,Ile⁴,Ile⁸](SII)-angiotensin (SII), a triply mutated angiotensin octapeptide that, in transfected cell systems, acts as a specific agonist for β-arrestin-mediated signaling, although not activating G protein-mediated signaling (8).Accordingly, in the studies described here, we set out to investigate the characteristics of activation of ERK in rat VSMCs that might be mediated through G protein as well as β-arrestin signaling. The results not only demonstrate the importance of β-arrestin-mediated signaling in ERK-mediated proliferative responses of these cells, but also shed new light on the molecular mechanisms and interrelationships between the β-arrestin and classical G protein-mediated activation of these pathways.     

Habitat use by yellowheads,Mohoua ochrocephala (Aves: Muscicapidae), in the Hawdon River Valley,Arthur’s Pass National Park. 2. Time budgets and foraging behaviour

Abstract

Instantaneous sampling was used to describe the ecological niche of yellowheads. Observations began during nesting in 1983 and continued until April 1984. Yellowheads spent on average 90% of their time foraging. As daylength decreased, an increasing proportion of time was spent foraging and a decreasing proportion of time was spent on social activities. When feeding nestlings, females spent significantly more time foraging than did males. Yellowheads spent 75% of their time in the upper understorey and the shaded canopy. There was no difference in the relative use of strata in the two canopy tree species, nor a sexual difference in the time spent in each strata. Yellowheads were entirely insectivorous. Prey items were recorded on 33 occasions; most were lepidopteran larvae. The most common foraging method was surface gleaning, most often on foliage and trunks. Time spent foraging on different substrates varied with tree diameter and tree species. Relative use of different foraging methods changed during the study, as did the types of substrates searched for prey and the proportion of time spent in different strata and at different heights in the forest. Presumably these changes were in response to variations in invertebrate availability.     

Genetic Inhibition of Phosphorylation of the Translation Initiation Factor eIF2α Does Not Block Aβ-Dependent Elevation of BACE1 and APP Levels or Reduce Amyloid Pathology in a Mouse Model of Alzheimer’s Disease

β-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) initiates the production of β-amyloid (Aβ), the major constituent of amyloid plaques in Alzheimer’s disease (AD). BACE1 is elevated ∼2–3 fold in AD brain and is concentrated in dystrophic neurites near plaques, suggesting BACE1 elevation is Aβ−dependent. Previously, we showed that phosphorylation of the translation initiation factor eIF2α de-represses translation of BACE1 mRNA following stress such as energy deprivation. We hypothesized that stress induced by Aβ might increase BACE1 levels by the same translational mechanism involving eIF2α phosphorylation. To test this hypothesis, we used three different genetic strategies to determine the effects of reducing eIF2α phosphorylation on Aβ-dependent BACE1 elevation in vitro and in vivo: 1) a two-vector adeno-associated virus (AAV) system to express constitutively active GADD34, the regulatory subunit of PP1c eIF2α phosphatase; 2) a non-phosphorylatable eIF2α S51A knockin mutation; 3) a BACE1-YFP transgene lacking the BACE1 mRNA 5′ untranslated region (UTR) required for eIF2α translational regulation. The first two strategies were used in primary neurons and 5XFAD transgenic mice, while the third strategy was employed only in 5XFAD mice. Despite very effective reduction of eIF2α phosphorylation in both primary neurons and 5XFAD brains, or elimination of eIF2α-mediated regulation of BACE1-YFP mRNA translation in 5XFAD brains, Aβ-dependent BACE1 elevation was not decreased. Additionally, robust inhibition of eIF2α phosphorylation did not block Aβ-dependent APP elevation in primary neurons, nor did it reduce amyloid pathology in 5XFAD mice. We conclude that amyloid-associated BACE1 elevation is not caused by translational de-repression via eIF2α phosphorylation, but instead appears to involve a post-translational mechanism. These definitive genetic results exclude a role for eIF2α phosphorylation in Aβ-dependent BACE1 and APP elevation. We suggest a vicious pathogenic cycle wherein Aβ42 toxicity induces peri-plaque BACE1 and APP accumulation in dystrophic neurites leading to exacerbated Aβ production and plaque progression.     

Susceptibility to Crohn’s disease is mediated by <Emphasis Type="Italic">KIR</Emphasis>2DL2/<Emphasis Type="Italic">KIR</Emphasis>2DL3 heterozygosity and the HLA-C ligand

In the present study, we investigated the relationship between the KIR loci and the genes encoding their HLA ligands and genetic susceptibility to Crohn’s disease (CD). Analyses of the interactions between KIR3DL1, KIR2DL1, KIR2DL2, and KIR2DL3 with their respective HLA ligands indicate that there is a protective effect for KIR2DL2 in the absence of its HLA ligand C1. Given that KIR2DL2 and KIR2DL3 segregate as alleles, we compared their genotypic distributions to expectations under Hardy–Weinberg Equilibrium (HWE) with regard to the HLA ligand C1 status. While all the genotypic distributions conform to expectations under HWE in controls, in C2 ligand homozygous cases there is significant deviation from HWE, with a reduction of KIR2DL2, KIR2DL3 heterozygotes. KIR2DL2, KIR2DL3 heterozygosity is the only genotypic combination that confers protection from CD. In addition to the protective effect (OR = 0.44, CI = 0.22–0.87; p = 0.018) observed in C2 ligand homozygotes, the KIR2DL2, KIR2DL3 genotype is predisposing (OR = 1.34, CI = 1.03–4.53; p = 0.031) in the presence of C1 ligand. A test for trend of HLA class I C ligand group genotypes with KIR2DL2, KIR2DL3 heterozygosity in cases and controls indicates that C1, C2 ligand group heterozygotes have an intermediate effect on predisposition. These results show for the first time that disease susceptibility may be related to heterozygosity at a specific KIR locus, and that HLA ligand genotype influences the relative effect of the KIR genotype.