The chondroitin sulfate A-binding site of the VAR2CSA protein involves multiple N-terminal domains

Malaria during pregnancy is a major health problem for African women. The disease is caused by Plasmodium falciparum malaria parasites, which accumulate in the placenta by adhering to chondroitin sulfate A (CSA). The interaction between infected erythrocytes and the placental receptor is mediated by a parasite expressed protein named VAR2CSA. A vaccine protecting pregnant women against placental malaria should induce antibodies inhibiting the interaction between VAR2CSA and CSA. Much effort has been put into defining the part of the 350 kDa VAR2CSA protein that is responsible for binding. It has been shown that full-length recombinant VAR2CSA binds specifically to CSA with high affinity, however to date no sub-fragment of VAR2CSA has been shown to interact with CSA with similar affinity or specificity. In this study, we used a biosensor technology to examine the binding properties of a panel of truncated VAR2CSA proteins. The experiments indicate that the core of the CSA-binding site is situated in three domains, DBL2X-CIDR(PAM) and a flanking domain, located in the N-terminal part of VAR2CSA. Furthermore, recombinant VAR2CSA subfragments containing this region elicit antibodies with high parasite adhesion blocking activity in animal immunization experiments.     

Chondroitin sulphate A (CSA)-binding of single recombinant Duffy-binding-like domains is not restricted to Plasmodium falciparum Erythrocyte Membrane Protein 1 expressed by CSA-binding parasites

Individuals living in areas with high Plasmodium falciparum transmission acquire immunity to malaria over time and adults have a markedly reduced risk of contracting severe disease. However, pregnant women constitute an important exception. Pregnancy-associated malaria is a major cause of mother and offspring morbidity, such as severe maternal anaemia and low birth-weight, and is characterised by selective accumulation of parasite-infected erythrocytes (IE) in the placenta. A P. falciparum protein named VAR2CSA, which belongs to the large P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1) family, enables the IE to bind chondroitin sulphate A (CSA) in the placenta. Knock-out studies have demonstrated the exclusive capacity of VAR2CSA to mediate IE binding to CSA, and it has been shown that four of the six Duffy-binding-like (DBL) domains of VAR2CSA have the ability to bind CSA in vitro. In this study, we confirm the CSA-binding of these DBL domains, however, the analysis of a number of DBL domains of a non-VAR2CSA origin shows that CSA-binding is not exclusively restricted to VAR2CSA DBL domains. Furthermore, we show that the VAR2CSA DBL domains as well as other DBL domains also bind heparan sulphate. These data explain a number of publications describing CSA-binding domains derived from PfEMP1 antigens not involved in placental adhesion. The data suggest that the ability of single domains to bind CSA does not predict the functional capacity of the whole PfEMP1 and raises doubt whether the CSA-binding domains of native VAR2CSA have been correctly identified.     

The specificity of the malarial VAR2CSA protein for chondroitin sulfate depends on 4-O-sulfation and ligand accessibility

Placental malaria infection is mediated by the binding of the malarial VAR2CSA protein to the placental glycosaminoglycan, chondroitin sulfate. Recombinant subfragments of VAR2CSA (rVAR2) have also been shown to bind specifically and with high affinity to cancer cells and tissues, suggesting the presence of a shared type of oncofetal chondroitin sulfate (ofCS) in the placenta and in tumors. However, the exact structure of ofCS and what determines the selective tropism of VAR2CSA remains poorly understood. In this study, ofCS was purified by affinity chromatography using rVAR2 and subjected to detailed structural analysis. We found high levels of N-acetylgalactosamine 4-O-sulfation (∼80–85%) in placenta- and tumor-derived ofCS. This level of 4-O-sulfation was also found in other tissues that do not support parasite sequestration, suggesting that VAR2CSA tropism is not exclusively determined by placenta- and tumor-specific sulfation. Here, we show that both placenta and tumors contain significantly more chondroitin sulfate moieties of higher molecular weight than other tissues. In line with this, CHPF and CHPF2, which encode proteins required for chondroitin polymerization, are significantly upregulated in most cancer types. CRISPR/Cas9 targeting of CHPF and CHPF2 in tumor cells reduced the average molecular weight of cell-surface chondroitin sulfate and resulted in a marked reduction of rVAR2 binding. Finally, utilizing a cell-based glycocalyx model, we showed that rVAR2 binding correlates with the length of the chondroitin sulfate chains in the cellular glycocalyx. These data demonstrate that the total amount and cellular accessibility of chondroitin sulfate chains impact rVAR2 binding and thus malaria infection.     

Structural insight into epitopes in the pregnancy-associated malaria protein VAR2CSA

Pregnancy-associated malaria is caused by Plasmodium falciparum malaria parasites binding specifically to chondroitin sulfate A in the placenta. This sequestration of parasites is a major cause of low birth weight in infants and anemia in the mothers. VAR2CSA, a polymorphic multi-domain protein of the PfEMP1 family, is the main parasite ligand for CSA binding, and identification of protective antibody epitopes is essential for VAR2CSA vaccine development. Attempts to determine the crystallographic structures of VAR2CSA or its domains have not been successful yet. In this study, we propose 3D models for each of the VAR2CSA DBL domains and we show that regions in the fold of VAR2CSA inter-domain 2 and a PfEMP1 CIDR domain seem to be homologous to the EBA-175 and Pk alpha-DBL fold. This suggests that ID2 could be a functional domain. We also identify regions of VAR2CSA present on the surface of native VAR2CSA by comparing reactivity of plasma containing anti-VAR2CSA antibodies in peptide array experiments before and after incubation with native VAR2CSA. By this method we identify conserved VAR2CSA regions targeted by antibodies that react with the native molecule expressed on infected erythrocytes. By mapping the data onto the DBL models we present evidence suggesting that the S1+S2 DBL sub-domains are generally surface-exposed in most domains, whereas the S3 sub-domains are less exposed in native VAR2CSA. These results comprise an important step towards understanding the structure of VAR2CSA on the surface of CSA-binding infected erythrocytes.     

Molecular architecture and domain arrangement of the placental malaria protein VAR2CSA suggests a model for carbohydrate binding

VAR2CSA is the placental-malaria–specific member of the antigenically variant Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family. It is expressed on the surface of Plasmodium falciparum-infected host red blood cells and binds to specific chondroitin-4-sulfate chains of the placental proteoglycan receptor. The functional ∼310 kDa ectodomain of VAR2CSA is a multidomain protein that requires a minimum 12-mer chondroitin-4-sulfate molecule for specific, high affinity receptor binding. However, it is not known how the individual domains are organized and interact to create the receptor-binding surface, limiting efforts to exploit its potential as an effective vaccine or drug target. Using small angle X-ray scattering and single particle reconstruction from negative-stained electron micrographs of the ectodomain and multidomain constructs, we have determined the structural architecture of VAR2CSA. The relative locations of the domains creates two distinct pores that can each accommodate the 12-mer of chondroitin-4-sulfate, suggesting a model for receptor binding. This model has important implications for understanding cytoadherence of infected red blood cells and potentially provides a starting point for developing novel strategies to prevent and/or treat placental malaria.     

Structural analysis of the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) intracellular domain reveals a conserved interaction epitope

Plasmodium falciparum-infected red blood cells adhere to endothelial cells, thereby obstructing the microvasculature. Erythrocyte adherence is directly associated with severe malaria and increased disease lethality, and it is mediated by the PfEMP1 family. PfEMP1 clustering in knob-like protrusions on the erythrocyte membrane is critical for cytoadherence, however the molecular mechanisms behind this system remain elusive. Here, we show that the intracellular domains of the PfEMP1 family (ATS) share a unique molecular architecture, which comprises a minimal folded core and extensive flexible elements. A conserved flexible segment at the ATS center is minimally restrained by the folded core. Yeast-two-hybrid data and a novel sequence analysis method suggest that this central segment contains a conserved protein interaction epitope. Interestingly, ATS in solution fails to bind the parasite knob-associated histidine-rich protein (KAHRP), an essential cytoadherence component. Instead, we demonstrate that ATS associates with PFI1780w, a member of the Plasmodium helical interspersed sub-telomeric (PHIST) family. PHIST domains are widespread in exported parasite proteins, however this is the first specific molecular function assigned to any variant of this family. We propose that PHIST domains facilitate protein interactions, and that the conserved ATS epitope may be targeted to disrupt the parasite cytoadherence system.     

Structural basis for binding of Plasmodium falciparum erythrocyte membrane protein 1 to chondroitin sulfate and placental tissue and the influence of protein polymorphisms on binding specificity

Chondroitin sulfate (CS) A is a key receptor for adhesion of Plasmodium falciparum-infected erythrocytes (IEs) in the placenta and can also mediate adhesion to microvascular endothelial cells. IEs that adhere to CSA express var2csa-type genes, which encode specific variants of the IE surface antigen P. falciparum erythrocyte membrane protein 1 (PfEMP1). We report direct binding of native PfEMP1, isolated from IEs and encoded by var2csa, to immobilized CSA. Binding of PfEMP1 was dependent on 4-O-sulfated disaccharides and glucuronic acid rather than iduronic acid, consistent with the specificity of intact IEs. Using immobilized CS oligosaccharides as neoglycolipid probes, the minimum chain length for direct binding of PfEMP1 was eight monosaccharide units. Similarly for IE adhesion to placental tissue there was a requirement for 4-O-sulfated GalNAc and glucuronic acid mixed with non-sulfated disaccharides; 6-O-sulfation interfered with the interaction between placental CSA and IEs. The minimum chain length for maximal inhibition of adhesion was 10 monosaccharide residues. Partially 4-O-sulfated CS oligosaccharides (45-55% sulfation) were highly effective inhibitors of placental adhesion (IC(50), 0.15 microg/ml) and may have potential for therapeutic development. We used defined P. falciparum isolates expressing different variants of var2csa in adhesion assays and found that there were isolate-specific differences in the preferred structural motifs for adhesion to CSA that correlated with polymorphisms in PfEMP1 encoded by var2csa-type genes. This may influence sites of IE sequestration or parasite virulence. These findings have significant implications for understanding the pathogenesis and biology of malaria, particularly during pregnancy, and the development of targeted interventions.     

Plasmodium falciparum: chondroitin sulfate A is the major receptor for adhesion of parasitized erythrocytes in the placenta

Plasmodium falciparum parasites that sequester in the placenta bind to the molecule chondroitin sulfate A (CSA). Women become resistant to malaria during pregnancy as they acquire antibodies that inhibit parasite adhesion to CSA, suggesting that a vaccine against placental malaria is feasible. Hyaluronic acid (HA) and non-immune IgG have also been proposed as receptors for P. falciparum adhesion in the placenta, but evidence for their roles is inconclusive. In this study, CSA, HA, and IgG were simultaneously assessed for their relative contributions to placental adhesion. Placental parasites collected in Tanzania uniformly adhered to the molecule CSA, and soluble CSA completely inhibited adhesion of most samples to placental cryosections. Three of 46 placental parasite samples also adhered to immobilized HA, but HA failed to inhibit adhesion of any placental parasites to placental cryosections. Similarly, non-immune IgG and protein A failed to inhibit adhesion of parasite samples to placental cryosection. P. falciparum adhesion in the placenta appears to be a non-redundant process that requires CSA as a receptor. Vaccines that elicit functional antibodies against CSA-binding parasites may confer resistance to pregnancy malaria.     

Plasmodium falciparum: Assessment of parasite-infected red blood cell binding to placental chondroitin proteoglycan and bovine tracheal chondroitin sulfate A

In pregnant women infected with Plasmodium falciparum, the infected red blood cells (IRBCs) sequester in placenta by binding to the chondroitin 4-sulfate (C4S) chains of low sulfated chondroitin sulfate proteoglycan (CSPG). Placental CSPG, the natural receptor for IRBC adherence in the placenta, is the ideal molecule for studying structural interactions in IRBC adhesion to C4S, adhesion inhibitory antibody responses, and identification of parasite adhesive protein(s). However, because of difficulty involved in purifying placental CSPG, the commercially available bovine tracheal chondroitin sulfate A (bCSA), a copolymer having structural features of both C4S and C6S, has been widely used. To determine the validity of bCSA for C4S-IRBC interaction studies, we comparatively evaluated the characteristics of IRBC binding to placental CSPG and bCSA using three commonly used parasite strains. The results indicate that, in all three parasites studied, the characteristics of IRBC binding to placental CSPG and bCSA are qualitatively similar, but the binding capacity with respect to both the number of IRBCs bound per unit area of coated surface and binding strength is significantly higher for CSPG than bCSA regardless of whether parasites were selected on CSPG or bCSA. These results demonstrate that placental CSPG is best suited for studying interactions between parasite adhesive protein(s) and C4S, and have implications in understanding C4S-IRBC structural interactions.     

Full-Length Recombinant Plasmodium falciparum VAR2CSA Binds Specifically to CSPG and Induces Potent Parasite Adhesion-Blocking Antibodies

Plasmodium falciparum malaria remains one of the world's leading causes of human suffering and poverty. Each year, the disease takes 1-3 million lives, mainly in sub-Saharan Africa. The adhesion of infected erythrocytes (IEs) to vascular endothelium or placenta is the key event in the pathogenesis of severe P. falciparum infection. In pregnant women, the parasites express a single and unique member of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family named VAR2CSA, which is associated with the ability of the IEs to adhere specifically to chondroitin sulphate A (CSA) in the placenta. Several Duffy-binding-like domains from VAR2CSA molecules have been shown in vitro to bind to CSA, but it has also been demonstrated that Duffy-binding-like domains from PfEMP1 proteins other than VAR2CSA can bind CSA. In addition, the specificity of the binding of VAR2CSA domains to glycosaminoglycans does not match that of VAR2CSA-expressing IEs. This has led to speculation that the domains of native VAR2CSA need to come together to form a specific binding site or that VAR2CSA might bind to CSA through a bridging molecule. Here, we describe the expression and purification of the complete extracellular region of VAR2CSA secreted at high yields from insect cells. Using surface plasmon resonance, we demonstrate that VAR2CSA alone binds with nanomolar affinity to human chondroitin sulphate proteoglycan and with significantly weaker affinity to other glycosaminoglycans, showing a specificity similar to that observed for IEs. Antibodies raised against full-length VAR2CSA completely inhibit recombinant VAR2CSA binding, as well as parasite binding to chondroitin sulphate proteoglycan. This is the first study to describe the successful production and functionality of a full-length PfEMP1. The specificity of the binding and anti-adhesion potency of induced IgG, together with high-yield production, encourages the use of full-length PfEMP1 in vaccine development strategies.     

Using Mutagenesis and Structural Biology to Map the Binding Site for the Plasmodium falciparum Merozoite Protein PfRh4 on the Human Immune Adherence Receptor

To survive and replicate within the human host, malaria parasites must invade erythrocytes. Invasion can be mediated by the P. falciparum reticulocyte-binding homologue protein 4 (PfRh4) on the merozoite surface interacting with complement receptor type 1 (CR1, CD35) on the erythrocyte membrane. The PfRh4 attachment site lies within the three N-terminal complement control protein modules (CCPs 1–3) of CR1, which intriguingly also accommodate binding and regulatory sites for the key complement activation-specific proteolytic products, C3b and C4b. One of these regulatory activities is decay-accelerating activity. Although PfRh4 does not impact C3b/C4b binding, it does inhibit this convertase disassociating capability. Here, we have employed ELISA, co-immunoprecipitation, and surface plasmon resonance to demonstrate that CCP 1 contains all the critical residues for PfRh4 interaction. We fine mapped by homologous substitution mutagenesis the PfRh4-binding site on CCP 1 and visualized it with a solution structure of CCPs 1–3 derived by NMR and small angle x-ray scattering. We cross-validated these results by creating an artificial PfRh4-binding site through substitution of putative PfRh4-interacting residues from CCP 1 into their homologous positions within CCP 8; strikingly, this engineered binding site had an ∼30-fold higher affinity for PfRh4 than the native one in CCP 1. These experiments define a candidate site on CR1 by which P. falciparum merozoites gain access to human erythrocytes in a non-sialic acid-dependent pathway of merozoite invasion.     

Plasmodium falciparum: worldwide sequence diversity and evolution of the malaria vaccine candidate merozoite surface protein-2 (MSP-2)

We examined patterns and putative mechanisms of sequence diversification in the merozoite surface protein-2 (MSP-2) of Plasmodium falciparum, a major dimorphic malaria vaccine candidate antigen, by analyzing 448 msp-2 alleles from all continents. We describe several nucleotide replacements, insertion and deletion events, frameshift mutations, and proliferations of repeat units that generate the extraordinary diversity found in msp-2 alleles. We discuss the role of positive selection exerted by naturally acquired type- and variant-specific immunity in maintaining the observed levels of polymorphism and suggest that this is the most likely explanation for the significant excess of nonsynonymous nucleotide replacements found in dimorphic msp-2 domains. Hybrid sequences created by meiotic recombination between alleles of different dimorphic types were observed in few (3.1%) isolates, mostly from Africa. We found no evidence for an extremely ancient origin of allelic dimorphism at the msp-2 locus, predating P. falciparum speciation, in contrast with recent findings for other surface malarial antigens.     

Structural Insights into the C1q Domain of Caprin-2 in Canonical Wnt Signaling

Previously, we have identified Caprin-2 as a new regulator in canonical Wnt signaling through a mechanism of facilitating LRP5/6 phosphorylation; moreover, we found that its C-terminal C1q-related domain (Cap2_CRD) is required for this process. Here, we determined the crystal structures of Cap2_CRD from human and zebrafish, which both associate as a homotrimer with calcium located at the symmetric center. Surprisingly, the calcium binding-deficient mutant exists as a more stable trimer than its wild-type counterpart. Further studies showed that this Caprin-2 mutant disabled in binding calcium maintains the activity of promoting LRP5/6 phosphorylation, whereas the mutations disrupting Cap2_CRD homotrimer did impair such activity. Together, our findings suggested that the C-terminal CRD domain of Caprin-2 forms a flexible homotrimer mediated by calcium and that such trimeric assembly is required for Caprin-2 to regulate canonical Wnt signaling.     

Adherence of Plasmodium falciparum infected erythrocytes to CHO-745 cells and inhibition of binding by protein A in the presence of human serum

Adhesion of erythrocytes infected with the malaria parasite Plasmodium falciparum to human host receptors is a process associated with severe malarial pathology. A number of in vitro cell lines are available as models for these adhesive processes, including Chinese hamster ovary (CHO) cells which express the placental adhesion receptor chondroitin-4-sulphate (CSA) on their surface. CHO-745 cells, a glycosaminoglycan-negative mutant CHO cell line lacking CSA and other reported P. falciparum adhesion receptors, are often used for recombinant expression of host receptors and for receptor binding studies. In this study we show that P. falciparum-infected erythrocytes can be easily selected for adhesion to an endogenous receptor on the surface of CHO-745 cells, bringing into question the validity of using these cells as a tool for P. falciparum adhesin expression studies. The adhesive interaction between CHO-745 cells and parasitized erythrocytes described here is not mediated by the known P. falciparum adhesion receptors CSA, CD36, or ICAM-1. However, we found that CHO-745-selected parasitized erythrocytes bind normal human IgM and that adhesion to CHO-745 cells is inhibited by protein A in the presence of serum, but not in its absence, indicating a non-specific inhibitory effect. Thus, protein A, which has been used as an inhibitor for a recently described interaction between infected erythrocytes and the placenta, may not be an appropriate in vitro inhibitor for understanding in vivo adhesive interactions.     

Solution structure of the phosphotyrosine binding (PTB) domain of human tensin2 protein in complex with deleted in liver cancer 1 (DLC1) peptide reveals a novel peptide binding mode

The protein deleted in liver cancer 1 (DLC1) interacts with the tensin family of focal adhesion proteins to play a role as a tumor suppressor in a wide spectrum of human cancers. This interaction has been proven to be crucial to the oncogenic inhibitory capacity and focal adhesion localization of DLC1. The phosphotyrosine binding (PTB) domain of tensin2 predominantly interacts with a novel site on DLC1, not the canonical NPXY motif. In this study, we characterized this interaction biochemically and determined the complex structure of tensin2 PTB domain with DLC1 peptide by NMR spectroscopy. Our HADDOCK-derived complex structure model elucidates the molecular mechanism by which tensin2 PTB domain recognizes DLC1 peptide and reveals a PTB-peptide binding mode that is unique in that peptide occupies the binding site opposite to the canonical NPXY motif interaction site with the peptide utilizing a non-canonical binding motif to bind in an extended conformation and that the N-terminal helix, which is unique to some Shc- and Dab-like PTB domains, is required for binding. Mutations of crucial residues defined for the PTB-DLC1 interaction affected the co-localization of DLC1 and tensin2 in cells and abolished DLC1-mediated growth suppression of hepatocellular carcinoma cells. This tensin2 PTB-DLC1 peptide complex with a novel binding mode extends the versatile binding repertoire of the PTB domains in mediating diverse cellular signaling pathways as well as provides a molecular and structural basis for better understanding the tumor-suppressive activity of DLC1 and tensin2.     

Temporal changes in the expression of protein phosphatase 1 and protein phosphatase 2A in proliferating and differentiating murine erythroleukaemia cells

Rhythmic changes in the expression of protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) were investigated during hexamethylene bisacetamide (HMBA) induced differentiation of murine erythroleukaemic (MEL) cells. Cell extracts were analysed by SDS-PAGE and western immunoblotting using specific antibodies. An immunospecific band of molecular mass 36 kDa (catalytic subunit) was detected for PP1. For PP2A, two immunospecific bands of 32 kDa (proteolytically cleaved catalytic subunit) and 36 kDa (catalytic subunit) were observed. Comparisons of proliferating and differentiating cells using only one time point showed no significant differences between mean values for the expression of the PP1 or PP2A enzyme proteins. This kind of analysis, implying that HMBA had little effect, proved misleading, as comparisons using multiple time points showed rhythmic patterns of protein expression which were modulated by the differentiating agent. The effects were complex affecting both the frequency and phasing of rhythms. The results add further support for the view that live cells are multi-oscillators and for the concept that differentiation depends on changes in temporal organization of complex autodynamic feedback loops and multiple interactions between control circuits performing in parallel. In particular, modulation of the dynamics of key proteins, such as PP1 and PP2A, may be a possible mechanism for controlling cellular function and reversing transformation in accordance with long standing theoretical and other experimental data.     

Ancient ubiquitous protein 1 (AUP1) localizes to lipid droplets and binds the E2 ubiquitin conjugase G2 (Ube2g2) via its G2 binding region

Lipid droplets (LDs), the major intracellular storage sites for neutral lipids, consist of a neutral lipid core surrounded by a phospholipid monolayer membrane. In addition to their function in lipid storage, LDs participate in lipid biosynthesis and recently were implicated in proteasomal protein degradation and autophagy. To identify components of the protein degradation machinery on LDs, we studied several candidates identified in previous LD proteome analyses. Here, we demonstrate that the highly conserved and broadly expressed ancient ubiquitous protein 1 (AUP1) localizes to LDs, where it integrates into the LD surface in a monotopic fashion with both termini facing the cytosol. AUP1 contains a C-terminal domain with strong homology to a domain known as G2BR, which binds E2 ubiquitin conjugases. We show that AUP1, by means of its G2BR domain, binds to Ube2g2. This binding is abolished by deletion or mutation of the G2BR domain, although the LD localization of AUP1 is not affected. The presence of the AUP1-Ube2g2 complex at LDs provides a direct molecular link between LDs and the cellular ubiquitination machinery.     

The functional cooperation of MAP1A heavy chain and light chain 2 in the binding of microtubules

Microtubule-associated protein 1A (MAP1A) is a high-molecular-weight protein that is comprised of a heavy chain and a light chain (LC2) and is widely distributed along the microtubules in both mature neurons and glial cells. To illustrate the interaction among the MAP1A heavy chain, light chain, and microtubule, we prepared DNA constructs with Myc-, EGFP-, or DsRed-tags for full-length MAP1A DNA expressing whole MAP1A protein, two domains of MAP1A heavy chain, and light chain. Distribution patterns of various MAP1A domains as well as their interactions with microtubules were monitored in a non-neuronal COS7 and a neuronal Neuro2A cells. Our data revealed that a complete MAP1A protein, which contains both heavy chain and LC2, could be colocalized with microtubule networks not only in Neuro2A cells but also in transfected COS7 cells. Filamentous structures failed to be visualized along microtubules in COS7 cells transfected with MAP1A heavy chain or LC2 alone. Whereas, after introducing MAP1A heavy chain with LC2 into COS7 cells, both heavy chain and LC2 could be colocalized with microtubules. From our functional analysis, both MAP1A and its LC2 could protect microtubules against the challenge of nacodazol. Data collected from yeast two-hybrid assays of various MAP1A domains confirmed that the interaction of LC2 and NH2-terminal of MAP1A heavy chain is important for microtubule binding. From our analysis of MAP1A functional domains, we suggest that interactions between MAP1A heavy chain and LC2 are critical for the binding of microtubules.     

Evolved to be active: sulfate ions define substrate recognition sites of CK2alpha and emphasise its exceptional role within the CMGC family of eukaryotic protein kinases

CK2alpha is the catalytic subunit of protein kinase CK2 and a member of the CMGC family of eukaryotic protein kinases like the cyclin-dependent kinases, the MAP kinases and glycogen-synthase kinase 3. We present here a 1.6 A resolution crystal structure of a fully active C-terminal deletion mutant of human CK2alpha liganded by two sulfate ions, and we compare this structure systematically with representative structures of related CMGC kinases. The two sulfate anions occupy binding pockets at the activation segment and provide the structural basis of the acidic consensus sequence S/T-D/E-X-D/E that governs substrate recognition by CK2. The anion binding sites are conserved among those CMGC kinases. In most cases they are neutralized by phosphorylation of a neighbouring threonine or tyrosine side-chain, which triggers conformational changes for regulatory purposes. CK2alpha, however, lacks both phosphorylation sites at the activation segment and structural plasticity. Here the anion binding sites are functionally changed from regulation to substrate recognition. These findings underline the exceptional role of CK2alpha as a constitutively active enzyme within a family of strictly controlled protein kinases.