Structural insights into collagen binding by platelet receptor glycoprotein VI.

Glycoprotein VI (GPVI) mediates collagen-induced platelet activation after vascular damage and is an important contributor to the onset of thrombosis, heart attack, and stroke. Animal models of thrombosis have identified GPVI as a promising target for antithrombotic therapy. Although for many years the crystal structure of GPVI has been known, the essential details of its interaction with collagen have remained elusive. Here, we present crystal structures of the GPVI ectodomain bound to triple-helical collagen peptides, which reveal a collagen-binding site across the ß-sheet of the D1 domain. Mutagenesis and binding studies confirm the observed binding site and identify Trp76, Arg38, and Glu40 as essential residues for binding to fibrillar collagens and collagen-related peptides (CRPs). GPVI binds a site on collagen comprising two collagen chains with the core formed by the sequence motif OGPOGP. Potent GPVI-binding peptides from Toolkit-III all contain OGPOGP; weaker binding peptides frequently contain a partial motif varying at either terminus. Alanine-scanning of peptide III-30 also identified two AGPOGP motifs that contribute to GPVI binding, but steric hindrance between GPVI molecules restricts the maximum binding capacity. We further show that no cooperative interactions could occur between two GPVI monomers binding to a stretch of (GPO)5 and that binding of ≥2 GPVI molecules to a fibril-embedded helix requires non-overlapping OGPOGP motifs. Our structure confirms the previously suggested similarity in collagen binding between GPVI and leukocyte-associated immunoglobulin-like receptor 1 (LAIR-1) but also indicates significant differences that may be exploited for the development of receptor-specific therapeutics.

Coronavirus hemagglutinin-esterase and spike proteins coevolve for functional balance and optimal virion avidity.

Human coronaviruses OC43 and HKU1 are respiratory pathogens of zoonotic origin that have gained worldwide distribution. OC43 apparently emerged from a bovine coronavirus (BCoV) spillover. All three viruses attach to 9-O-acetylated sialoglycans via spike protein S with hemagglutinin-esterase (HE) acting as a receptor-destroying enzyme. In BCoV, an HE lectin domain promotes esterase activity toward clustered substrates. OC43 and HKU1, however, lost HE lectin function as an adaptation to humans. Replaying OC43 evolution, we knocked out BCoV HE lectin function and performed forced evolution-population dynamics analysis. Loss of HE receptor binding selected for second-site mutations in S, decreasing S binding affinity by orders of magnitude. Irreversible HE mutations led to cooperativity in virus swarms with low-affinity S minority variants sustaining propagation of high-affinity majority phenotypes. Salvageable HE mutations induced successive second-site substitutions in both S and HE. Apparently, S and HE are functionally interdependent and coevolve to optimize the balance between attachment and release. This mechanism of glycan-based receptor usage, entailing a concerted, fine-tuned activity of two envelope protein species, is unique among CoVs, but reminiscent of that of influenza A viruses. Apparently, general principles fundamental to virion-sialoglycan interactions prompted convergent evolution of two important groups of human and animal pathogens.

Human coronaviruses OC43 and HKU1 bind to 9-O-acetylated sialic acids via a conserved receptor-binding site in spike protein domain A.

Human betacoronaviruses OC43 and HKU1 are endemic respiratory pathogens and, while related, originated from independent zoonotic introductions. OC43 is in fact a host-range variant of the species Betacoronavirus-1, and more closely related to bovine coronavirus (BCoV)-its presumptive ancestor-and porcine hemagglutinating encephalomyelitis virus (PHEV). The ß1-coronaviruses (ß1CoVs) and HKU1 employ glycan-based receptors carrying 9-O-acetylated sialic acid (9-O-Ac-Sia). Receptor binding is mediated by spike protein S, the main determinant of coronavirus host specificity. For BCoV, a crystal structure for the receptor-binding domain S1A is available and for HKU1 a cryoelectron microscopy structure of the complete S ectodomain. However, the location of the receptor-binding site (RBS), arguably the single-most important piece of information, is unknown. Here we solved the 3.0-Å crystal structure of PHEV S1A We then took a comparative structural analysis approach to map the ß1CoV S RBS, using the general design of 9-O-Ac-Sia-binding sites as blueprint, backed-up by automated ligand docking, structure-guided mutagenesis of OC43, BCoV, and PHEV S1A, and infectivity assays with BCoV-S-pseudotyped vesicular stomatitis viruses. The RBS is not exclusive to OC43 and related animal viruses, but is apparently conserved and functional also in HKU1 S1A The binding affinity of the HKU1 S RBS toward short sialoglycans is significantly lower than that of OC43, which we attribute to differences in local architecture and accessibility, and which may be indicative for differences between the two viruses in receptor fine-specificity. Our findings challenge reports that would map the OC43 RBS elsewhere in S1A and that of HKU1 in domain S1B.

Structure and flexibility of the extracellular region of the PirB receptor.

Murine paired immunoglobulin receptor B (PirB) and its human ortholog leukocyte immunoglobulin-like receptor B2 (LILRB2) are widely expressed inhibitory receptors that interact with a diverse set of extracellular ligands and exert functions ranging from down-regulation of immune responses to inhibition of neuronal growth. However, structural information that could shed light on how PirB interacts with its ligands is lacking. Here, we report crystal structures of the PirB ectodomain; the first full ectodomain structure for a LILR family member, at 3.3-4.5 Å resolution. The structures reveal that PirB's six Ig-like domains are arranged at acute angles, similar to the structures of leukocyte immunoglobulin-like receptor (LILR) and killer-cell immunoglobulin-like receptor (KIR). We observe that this regular arrangement is followed throughout the ectodomain, resulting in an extended zigzag conformation. In two out of the five structures reported here, the repeating zigzag is broken by the first domain that can adopt two alternative orientations. Quantitative binding experiments revealed a 9 µm dissociation constant for PirB-myelin-associated glycoprotein (MAG) ectodomain interactions. Taken together, these structural findings and the observed PirB-MAG interactions are compatible with a model for intercellular signaling in which the PirB extracellular domains, which point away from the cell surface, enable interaction with ligands in trans.

Rare variants in GP1BB are responsible for autosomal dominant macrothrombocytopenia.

The von Willebrand receptor complex, which is composed of the glycoproteins Ibα, Ibß, GPV, and GPIX, plays an essential role in the earliest steps in hemostasis. During the last 4 decades, it has become apparent that loss of function of any 1 of 3 of the genes encoding these glycoproteins (namely, GP1BA, GP1BB, and GP9) leads to autosomal recessive macrothrombocytopenia complicated by bleeding. A small number of variants in GP1BA have been reported to cause a milder and dominant form of macrothrombocytopenia, but only 2 tentative reports exist of such a variant in GP1BB By analyzing data from a collection of more than 1000 genome-sequenced patients with a rare bleeding and/or platelet disorder, we have identified a significant association between rare monoallelic variants in GP1BB and macrothrombocytopenia. To strengthen our findings, we sought further cases in 2 additional collections in the United Kingdom and Japan. Across 18 families exhibiting phenotypes consistent with autosomal dominant inheritance of macrothrombocytopenia, we report on 27 affected cases carrying 1 of 9 rare variants in GP1BB.

Coronavirus receptor switch explained from the stereochemistry of protein-carbohydrate interactions and a single mutation.

Hemagglutinin-esterases (HEs) are bimodular envelope proteins of orthomyxoviruses, toroviruses, and coronaviruses with a carbohydrate-binding "lectin" domain appended to a receptor-destroying sialate-O-acetylesterase ("esterase"). In concert, these domains facilitate dynamic virion attachment to cell-surface sialoglycans. Most HEs (type I) target 9-O-acetylated sialic acids (9-O-Ac-Sias), but one group of coronaviruses switched to using 4-O-Ac-Sias instead (type II). This specificity shift required quasisynchronous adaptations in the Sia-binding sites of both lectin and esterase domains. Previously, a partially disordered crystal structure of a type II HE revealed how the shift in lectin ligand specificity was achieved. How the switch in esterase substrate specificity was realized remained unresolved, however. Here, we present a complete structure of a type II HE with a receptor analog in the catalytic site and identify the mutations underlying the 9-O- to 4-O-Ac-Sia substrate switch. We show that (i) common principles pertaining to the stereochemistry of protein-carbohydrate interactions were at the core of the transition in lectin ligand and esterase substrate specificity; (ii) in consequence, the switch in O-Ac-Sia specificity could be readily accomplished via convergent intramolecular coevolution with only modest architectural changes in lectin and esterase domains; and (iii) a single, inconspicuous Ala-to-Ser substitution in the catalytic site was key to the emergence of the type II HEs. Our findings provide fundamental insights into how proteins "see" sugars and how this affects protein and virus evolution.

Molecular basis determining species specificity for TLR2 inhibition by staphylococcal superantigen-like protein 3 (SSL3).

Staphylococcus aureus is a versatile opportunistic pathogen, causing disease in human and animal species. Its pathogenicity is linked to the ability of S. aureus to secrete immunomodulatory molecules. These evasion proteins bind to host receptors or their ligands, resulting in inhibitory effects through high affinity protein-protein interactions. Staphylococcal evasion molecules are often species-specific due to differences in host target proteins between species. We recently solved the crystal structure of murine TLR2 in complex with immunomodulatory molecule staphylococcal superantigen-like protein 3 (SSL3), which revealed the essential residues within SSL3 for TLR2 inhibition. In this study we aimed to investigate the molecular basis of the interaction on the TLR2 side. The SSL3 binding region on murine TLR2 was compared to that of other species through sequence alignment and homology modeling, which identified interspecies differences. To examine whether this resulted in altered SSL3 activity on the corresponding TLR2s, bovine, equine, human, and murine TLR2 were stably expressed in HEK293T cells and the ability of SSL3 to inhibit TLR2 was assessed. We found that SSL3 was unable to inhibit bovine TLR2. Subsequent loss and gain of function mutagenesis showed that the lack of inhibition is explained by the absence of two tyrosine residues in bovine TLR2 that play a prominent role in the SSL3-TLR2 interface. We found no evidence for the existence of allelic SSL3 variants that have adapted to the bovine host. Thus, within this paper we reveal the molecular determinants of the TLR2-SSL3 interaction which adds to our understanding of staphylococcal host specificity.

Structural basis for inhibition of TLR2 by staphylococcal superantigen-like protein 3 (SSL3).

Toll-like receptors (TLRs) are crucial in innate recognition of invading micro-organisms and their subsequent clearance. Bacteria are not passive bystanders and have evolved complex evasion mechanisms. Staphylococcus aureus secretes a potent TLR2 antagonist, staphylococcal superantigen-like protein 3 (SSL3), which prevents receptor stimulation by pathogen-associated lipopeptides. Here, we present crystal structures of SSL3 and its complex with TLR2. The structure reveals that formation of the specific inhibitory complex is predominantly mediated by hydrophobic contacts between SSL3 and TLR2 and does not involve interaction of TLR2-glycans with the conserved Lewis(X) binding site of SSL3. In the complex, SSL3 partially covers the entrance to the lipopeptide binding pocket in TLR2, reducing its size by ∼50%. We show that this is sufficient to inhibit binding of agonist Pam2CSK4 effectively, yet allows SSL3 to bind to an already formed TLR2-Pam2CSK4 complex. The binding site of SSL3 overlaps those of TLR2 dimerization partners TLR1 and TLR6 extensively. Combined, our data reveal a robust dual mechanism in which SSL3 interferes with TLR2 activation at two stages: by binding to TLR2, it blocks ligand binding and thus inhibits activation. Second, by interacting with an already formed TLR2-lipopeptide complex, it prevents TLR heterodimerization and downstream signaling.

Implications for collagen I chain registry from the structure of the collagen von Willebrand factor A3 domain complex.

Fibrillar collagens, the most abundant proteins in the vertebrate body, are involved in a plethora of biological interactions. Plasma protein von Willebrand factor (VWF) mediates adhesion of blood platelets to fibrillar collagen types I, II, and III, which is essential for normal haemostasis. High affinity VWF-binding sequences have been identified in the homotrimeric collagen types II and III, however, it is unclear how VWF recognizes the heterotrimeric collagen type I, the superstructure of which is unknown. Here we present the crystal structure of VWF domain A3 bound to a collagen type III-derived homotrimeric peptide. Our structure reveals that VWF-A3 interacts with all three collagen chains and binds through conformational selection to a sequence that is one triplet longer than was previously appreciated from platelet and VWF binding studies. The VWF-binding site overlaps those of SPARC (also known as osteonectin) and discodin domain receptor 2, but is more extended and shifted toward the collagen amino terminus. The observed collagen-binding mode of VWF-A3 provides direct structural constraints on collagen I chain registry. A VWF-binding site can be generated from the sequences RGQAGVMF, present in the two α1(I) chains, and RGEOGNIGF, in the unique α2(I) chain, provided that α2(I) is in the middle or trailing position. Combining these data with previous structural data on integrin binding to collagen yields strong support for the trailing position of the α2(I) chain, shedding light on the fundamental and long-standing question of the collagen I chain registry.

The murine coronavirus hemagglutinin-esterase receptor-binding site: a major shift in ligand specificity through modest changes in architecture.

The hemagglutinin-esterases (HEs), envelope glycoproteins of corona-, toro- and orthomyxoviruses, mediate reversible virion attachment to O-acetylated sialic acids (O-Ac-Sias). They do so through concerted action of distinct receptor-binding ("lectin") and receptor-destroying sialate O-acetylesterase ("esterase") domains. Most HEs target 9-O-acetylated Sias. In one lineage of murine coronaviruses, however, HE esterase substrate and lectin ligand specificity changed dramatically as these viruses evolved to use 4-O-acetylated Sias instead. Here we present the crystal structure of the lectin domain of mouse hepatitis virus (MHV) strain S HE, resolved both in its native state and in complex with a receptor analogue. The data show that the shift from 9-O- to 4-O-Ac-Sia receptor usage primarily entailed a change in ligand binding topology and, surprisingly, only modest changes in receptor-binding site architecture. Our findings illustrate the ease with which viruses can change receptor-binding specificity with potential consequences for host-, organ and/or cell tropism, and for pathogenesis.

When less becomes more: optimization of protein expression in HEK293-EBNA1 cells using plasmid titration - a case study for NLRs.

Transient transfection of the human HEK293-EBNA1 cell line using polyethyleneimine is widely adopted for recombinant protein production. Whereas high expression of many targets is achieved, purification yields of some highly expressed proteins remain low due to aggregation. We hypothesized that for these proteins the expression rates achieved at standard transfection conditions are too high, causing an overload of the protein folding machinery. Here we present plasmid titration as an efficient method to vary expression rates for the optimization of soluble protein expression. In plasmid titration a dilution series of expression vector mixed with dummy plasmid is transfected in small scale cultures. Application to GFP shows that plasmid titration achieves a wide range of expression levels while maintaining high transfection efficiencies even at 500-fold plasmid dilution. Application of plasmid titration to selected Nod-like receptors (NLRs), which at standard conditions are highly expressed but poorly soluble, delays the onset of NLR aggregation and improves cell viability and the buildup of biomass. The amount of soluble protein depends on the combination of dilution factor and harvest day in a protein specific manner. For NOD1 50-fold plasmid dilution increases the amount of soluble protein approximately 5-fold. Due to its association with chaperones at all dilution factors tested we were unable to purify NOD1 to homogeneity. For NLRC4, which did not associate with chaperones, 10-fold plasmid dilution increased the purification yield 2-fold. This improvement, obtained with minimal effort due to the simplicity of the method, shows that reducing total expression may increase soluble protein yield.

Formation and structure of a NAIP5-NLRC4 inflammasome induced by direct interactions with conserved N- and C-terminal regions of flagellin.

The NOD-like receptors NAIP5 and NLRC4 play an essential role in the innate immune response to the bacterial tail protein flagellin. Upon flagellin detection, NAIP5 and NLRC4 form a hetero-oligomeric inflammasome that induces caspase-1-dependent cell death. So far, both the mechanism of formation of the NAIP5-NLRC4 inflammasome and its structure are poorly understood. In this study we combine inflammasome reconstitution in HEK293 cells, purification of inflammasome components, and negative stain electron microscopy to address these issues. We find that a Salmonella typhimurium flagellin fragment comprising the D0 domain and the neighboring spoke region is able to co-precipitate NAIP5 and induce formation of the NAIP5-NLRC4 inflammasome. Comparison with smaller fragments indicates that flagellin recognition is mediated by its C-terminal residues as well as the spoke region. We reconstitute the inflammasome from purified flagellin, NAIP5, and NLRC4, thus proving that no other cellular components are required for its formation. Electron micrographs of the purified inflammasome provide unprecedented insight into its architecture, revealing disk-like complexes consisting of 11 or 12 protomers in which NAIP5 and NLRC4 appear to occupy equivalent positions. On the basis of our data, we propose a model for inflammasome formation wherein direct interaction of flagellin with a single NAIP5 induces the recruitment and progressive incorporation of NLRC4, resulting in the formation of a hetero-oligomeric inflammasome.

Crystal structure and collagen-binding site of immune inhibitory receptor LAIR-1: unexpected implications for collagen binding by platelet receptor GPVI.

Leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1), one of the most widely spread immune receptors, attenuates immune cell activation when bound to specific sites in collagen. The collagen-binding domain of LAIR-1 is homologous to that of glycoprotein VI (GPVI), a collagen receptor crucial for platelet activation. Because LAIR-1 and GPVI also display overlapping collagen-binding specificities, a common structural basis for collagen recognition would appear likely. Therefore, it is crucial to gain insight into the molecular interaction of both receptors with their ligand to prevent unwanted cross-reactions during therapeutic intervention. We determined the crystal structure of LAIR-1 and mapped its collagen-binding site by nuclear magnetic resonance (NMR) titrations and mutagenesis. Our data identify R59, E61, and W109 as key residues for collagen interaction. These residues are strictly conserved in LAIR-1 and GPVI alike; however, they are located outside the previously proposed GPVI collagen-binding site. Our data provide evidence for an unanticipated mechanism of collagen recognition common to LAIR-1 and GPVI. This fundamental insight will contribute to the exploration of specific means of intervention in collagen-induced signaling in immunity and hemostasis.

Factor B structure provides insights into activation of the central protease of the complement system.

Factor B is the central protease of the complement system of immune defense. Here, we present the crystal structure of human factor B at 2.3-A resolution, which reveals how the five-domain proenzyme is kept securely inactive. The canonical activation helix of the Von Willebrand factor A (VWA) domain is displaced by a helix from the preceding domain linker. The two helices conformationally link the scissile-activation peptide and the metal ion-dependent adhesion site required for binding of the ligand C3b. The data suggest that C3b binding displaces the three N-terminal control domains and reshuffles the two central helices. Reshuffling of the helices releases the scissile bond for final proteolytic activation and generates a new interface between the VWA domain and the serine protease domain. This allosteric mechanism is crucial for tight regulation of the complement-amplification step in the immune response.

Structural basis for ligand and substrate recognition by torovirus hemagglutinin esterases.

Hemagglutinin esterases (HEs), closely related envelope glycoproteins in influenza C and corona- and toroviruses, mediate reversible attachment to O-acetylated sialic acids (Sias). They do so by acting both as lectins and as receptor-destroying enzymes, functions exerted by separate protein domains. HE divergence was accompanied by changes in quaternary structure and in receptor and substrate specificity. The selective forces underlying HE diversity and the molecular basis for Sia specificity are poorly understood. Here we present crystal structures of porcine and bovine torovirus HEs in complex with receptor analogs. Torovirus HEs form homodimers with sialate-O-acetylesterase domains almost identical to corresponding domains in orthomyxo- and coronavirus HEs, but with unique lectin sites. Structure-guided biochemical analysis of the esterase domains revealed that a functionally, but not structurally conserved arginine-Sia carboxylate interaction is critical for the binding and positioning of glycosidically bound Sias in the catalytic pocket. Although essential for efficient de-O-acetylation of Sias, this interaction is not required for catalysis nor does it affect substrate specificity. In fact, the distinct preference of the porcine torovirus enzyme for 9-mono- over 7,9-di-O-acetylated Sias can be explained from a single-residue difference with HEs of more promiscuous specificity. Apparently, esterase and lectin pockets coevolved; also the porcine torovirus HE receptor-binding site seems to have been designed to use 9-mono- and exclude di-O-acetylated Sias, possibly as an adaptation to replication in swine. Our findings shed light on HE evolution and provide fundamental insight into mechanisms of substrate binding, substrate recognition, and receptor selection in this important class of virion proteins.

Structures of complement component C3 provide insights into the function and evolution of immunity.

The mammalian complement system is a phylogenetically ancient cascade system that has a major role in innate and adaptive immunity. Activation of component C3 (1,641 residues) is central to the three complement pathways and results in inflammation and elimination of self and non-self targets. Here we present crystal structures of native C3 and its final major proteolytic fragment C3c. The structures reveal thirteen domains, nine of which were unpredicted, and suggest that the proteins of the alpha2-macroglobulin family evolved from a core of eight homologous domains. A double mechanism prevents hydrolysis of the thioester group, essential for covalent attachment of activated C3 to target surfaces. Marked conformational changes in the alpha-chain, including movement of a critical interaction site through a ring formed by the domains of the beta-chain, indicate an unprecedented, conformation-dependent mechanism of activation, regulation and biological function of C3.

Structure of coronavirus hemagglutinin-esterase offers insight into corona and influenza virus evolution.

The hemagglutinin-esterases (HEs) are a family of viral envelope glycoproteins that mediate reversible attachment to O-acetylated sialic acids by acting both as lectins and as receptor-destroying enzymes (RDEs). Related HEs occur in influenza C, toro-, and coronaviruses, apparently as a result of relatively recent lateral gene transfer events. Here, we report the crystal structure of a coronavirus (CoV) HE in complex with its receptor. We show that CoV HE arose from an influenza C-like HE fusion protein (HEF). In the process, HE was transformed from a trimer into a dimer, whereas remnants of the fusion domain were adapted to establish novel monomer-monomer contacts. Whereas the structural design of the RDE-acetylesterase domain remained unaltered, the HE receptor-binding domain underwent remodeling to such extent that the ligand is now bound in opposite orientation. This is surprising, because the architecture of the HEF site was preserved in influenza A HA over a much larger evolutionary distance, a switch in receptor specificity and extensive antigenic variation notwithstanding. Apparently, HA and HEF are under more stringent selective constraints than HE, limiting their exploration of alternative binding-site topologies. We attribute the plasticity of the CoV HE receptor-binding site to evolutionary flexibility conferred by functional redundancy between HE and its companion spike protein S. Our findings offer unique insights into the structural and functional consequences of independent protein evolution after interviral gene exchange and open potential avenues to broad-spectrum antiviral drug design.

Crystal structures of TAFI elucidate the inactivation mechanism of activated TAFI: a novel mechanism for enzyme autoregulation.

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a pro-metallocarboxypeptidase that can be proteolytically activated (TAFIa). TAFIa is unique among carboxypeptidases in that it spontaneously inactivates with a short half-life, a property that is crucial for its role in controlling blood clot lysis. We studied the intrinsic instability of TAFIa by solving crystal structures of TAFI, a TAFI inhibitor (GEMSA) complex and a quadruple TAFI mutant (70-fold more stable active enzyme). The crystal structures show that TAFIa stability is directly related to the dynamics of a 55-residue segment (residues 296-350) that includes residues of the active site wall. Dynamics of this flap are markedly reduced by the inhibitor GEMSA, a known stabilizer of TAFIa, and stabilizing mutations. Our data provide the structural basis for a model of TAFI auto-regulation: in zymogen TAFI the dynamic flap is stabilized by interactions with the activation peptide. Release of the activation peptide increases dynamic flap mobility and in time this leads to conformational changes that disrupt the catalytic site and expose a cryptic thrombin-cleavage site present at Arg302. This represents a novel mechanism of enzyme control that enables TAFI to regulate its activity in plasma in the absence of specific inhibitors.

Erythrocytosis associated with a novel missense mutation in the HIF2A gene.

The ERYTHROPOIETIN (EPO) gene is regulated by the transcription factor Hypoxia Inducible Factor-alpha (HIF-alpha). In this pathway, Prolyl Hydroxylase Domain protein 2 (PHD2) hydroxylates two prolyl residues in HIF-alpha, which in turn promotes HIF-alpha degradation by the von Hippel Lindau (VHL) protein. Evidence that HIF-2alpha is the important isoform for EPO regulation in humans comes from the recent observation that mutations in the HIF2A gene are associated with cases of erythrocytosis. We report here a new erythrocytosis-associated mutation, p.Asp539Glu, in the HIF2A gene. Similar to all reported cases, the affected residue is in close vicinity and C-terminal to the primary hydroxylation site in HIF-2alpha, Pro531. This mutation, however, is notable in producing a rather subtle amino acid substitution. Nonetheless, we find that this mutation compromises binding of HIF-2alpha to both PHD2 and VHL, and we propose that this mutation is the cause of erythrocytosis in this individual.

Fifteen novel mutations in PKLR associated with pyruvate kinase (PK) deficiency: structural implications of amino acid substitutions in PK.

Pyruvate kinase (PK) deficiency is a rare disease but an important cause of hereditary nonspherocytic hemolytic anemia. The disease is caused by mutations in the PKLR gene and shows a marked variability in clinical expression. We report on the molecular characterization of 38 PK-deficient patients from 35 unrelated families. Twenty-nine different PKLR mutations were detected, of which 15 are reported here for the first time. Two novel deletions are reported: c.142_159del18 is the largest in-frame deletion described thus far and predicts the loss of six consecutive amino acids (p.Thr48_Thr53del) in the N-terminal domain of red blood cell PK. The other deletion removes nearly 1.5 kb of genomic DNA sequence (c.1618+37_2064del1477) and is one of a few large deletional mutants in PKLR. In addition, 13 novel point mutations were identified: one nonsense mutant, p.Arg488X, and 12 missense mutations, predicting the substitution of a single amino acid: p.Arg40Trp, p.Leu73Pro, p.Ile90Asn, p.Gly111Arg, p.Ala154Thr, p.Arg163Leu, p.Gly165Val, p.Leu272Val, p.Ile310Asn, p.Val320Leu, p.Gly358Glu, and p.Leu374Pro. We used the three-dimensional (3D) structure of recombinant human tetrameric PK to evaluate the protein structural context of the affected residues. In addition, in selected patients red blood cell PK antigen levels were measured by enzyme-linked immunosorbent assay (ELISA). Collectively, the results provided us with a rationale for the observed enzyme deficiency and contribute to both a better understanding of the genotype-to-phenotype correlation in PK deficiency as well as the enzyme's structure and function.