A conserved arginine within the αC-helix of Erk1/2 is a latch of autoactivation and of oncogenic capabilities.

Eukaryotic protein kinases (EPKs) adopt an active conformation following phosphorylation of a particular activation loop residue. Most EPKs spontaneously autophosphorylate this residue. While structure-function relationships of the active conformation are essentially understood, those of the "prone-to-autophosphorylate" conformation are unclear. Here, we propose that a site within the αC-helix of EPKs, occupied by Arg in the mitogen-activated protein kinase (MAPK) Erk1/2 (Arg84/65), impacts spontaneous autophosphorylation. MAPKs lack spontaneous autoactivation, but we found that converting Arg84/65 of Erk1/2 to various residues enables spontaneous autophosphorylation. Furthermore, Erk1 molecules mutated in Arg84 are oncogenic. Arg84/65 thus obstructs the adoption of the "prone-to-autophosphorylate" conformation. All MAPKs harbor an Arg that is equivalent to Arg84/65 of Erks, whereas Arg is rarely found at the equivalent position in other EPKs. We observed that Arg84/65 of Erk1/2 interacts with the DFG motif, suggesting that autophosphorylation may be inhibited by the Arg84/65-DFG interactions. Erk1/2s mutated in Arg84/65 autophosphorylate not only the TEY motif, known as critical for catalysis, but also on Thr207/188. Our MS/MS analysis revealed that a large proportion of the Erk2R65H population is phosphorylated on Thr188 or on Tyr185 + Thr188, and a small fraction is phosphorylated on the TEY motif. No molecules phosphorylated on Thr183 + Thr188 were detected. Thus, phosphorylation of Thr183 and Thr188 is mutually exclusive suggesting that not only TEY-phosphorylated molecules are active but perhaps also those phosphorylated on Tyr185 + Thr188. The effect of mutating Arg84/65 may mimic a physiological scenario in which allosteric effectors cause Erk1/2 activation by autophosphorylation.

The bacterial metalloprotease NleD selectively cleaves mitogen-activated protein kinases that have high flexibility in their activation loop.

Microbial pathogens often target the host mitogen-activated protein kinase (MAPK) network to suppress host immune responses. We previously identified a bacterial type III secretion system effector, termed NleD, a metalloprotease that inactivates MAPKs by specifically cleaving their activation loop. Here, we show that NleDs form a growing family of virulence factors harbored by human and plant pathogens as well as insect symbionts. These NleDs disable specifically Jun N-terminal kinases (JNKs) and p38s that are required for host immune response, whereas extracellular signal-regulated kinase (ERK), which is essential for host cell viability, remains intact. We investigated the mechanism that makes ERK resistant to NleD cleavage. Biochemical and structural analyses revealed that NleD exclusively targets activation loops with high conformational flexibility. Accordingly, NleD cleaved the flexible loops of JNK and p38 but not the rigid loop of ERK. Our findings elucidate a compelling mechanism of native substrate proteolysis that is promoted by entropy-driven specificity. We propose that such entropy-based selectivity is a general attribute of proteolytic enzymes.

Expression, purification and crystallization of CLK1 kinase - A potential target for antiviral therapy.

Cdc-like kinase 1 (CLK1) is a dual-specificity kinase capable of autophosphorylation on tyrosine residues and Ser/Thr phosphorylation of its substrates. CLK1 belongs to the CLK kinase family that regulates alternative splicing through phosphorylation of serine-arginine rich (SR) proteins. Recent studies have demonstrated that CLK1 has an important role in the replication of influenza A and chikungunya viruses. Furthermore, CLK1 was found to be relevant for the replication of HIV-1 and the West Nile virus, making CLK1 an interesting cellular candidate for the development of a host-directed antiviral therapy that might be efficient for treatment of newly emerging viruses. We describe here our attempts and detailed procedures to obtain the recombinant kinase domain of CLK1 in suitable amounts for crystallization in complex with specific inhibitors. The key solution for the reproducibility of crystals resides in devising and refining expression and purification protocols leading to homogeneous protein. Co-expression of CLK1 with λ-phosphatase and careful purification has yielded crystals of CLK1 complexed with the KH-CB19 inhibitor that diffracted to 1.65 Å. These results paved the path to the screening of more structures of CLK1 complexed compounds, leading to further optimization of their inhibitory activity. Moreover, since kinases are desired targets in numerous pathologies, the approach we report here, the co-expression of kinases with λ-phosphatase, previously used in other kinases, can be adopted as a general protocol in numerous kinase targets for obtaining reproducible and homogenic non-phosphorylated (inactive) forms suitable for biochemical and structural studies thus facilitating the development of novel inhibitors.

Distinctive ligand-binding specificities of tandem PA14 biomass-sensory elements from Clostridium thermocellum and Clostridium clariflavum.

Cellulolytic clostridia use a highly efficient cellulosome system to degrade polysaccharides. To regulate genes encoding enzymes of the multi-enzyme cellulosome complex, certain clostridia contain alternative sigma I (σI ) factors that have cognate membrane-associated anti-σI factors (RsgIs) which act as polysaccharide sensors. In this work, we analyzed the structure-function relationship of the extracellular sensory elements of Clostridium (Ruminiclostridium) thermocellum and Clostridium clariflavum (RsgI3 and RsgI4, respectively). These elements were selected for comparison, as each comprised two tandem PA14-superfamily motifs. The X-ray structures of the PA14 modular dyads from the two bacterial species were determined, both of which showed a high degree of structural and sequence similarity, although their binding preferences differed. Bioinformatic approaches indicated that the DNA sequence of promoter of sigI/rsgI operons represents a strong signature, which helps to differentiate binding specificity of the structurally similar modules. The σI4 -dependent C. clariflavum promoter sequence correlates with binding of RsgI4_PA14 to xylan and was identified in genes encoding xylanases, whereas the σI3 -dependent C. thermocellum promoter sequence correlates with RsgI3_PA14 binding to pectin and regulates pectin degradation-related genes. Structural similarity between clostridial PA14 dyads to PA14-containing proteins in yeast helped identify another crucial signature element: the calcium-binding loop 2 (CBL2), which governs binding specificity. Variations in the five amino acids that constitute this loop distinguish the pectin vs xylan specificities. We propose that the first module (PA14A ) is dominant in directing the binding to the ligand in both bacteria. The two X-ray structures of the different PA14 dyads represent the first reported structures of tandem PA14 modules.

Nuclear ERK Translocation is Mediated by Protein Kinase CK2 and Accelerated by Autophosphorylation.

BACKGROUND/AIMS: The extracellular signal-regulated kinases (ERK) 1 and 2 (ERK1/2) are members of the mitogen-activated protein kinase (MAPK) family. Upon stimulation, these kinases translocate from the cytoplasm to the nucleus, where they induce physiological processes such as proliferation and differentiation. The mechanism of translocation of this kinase involves phosphorylation of two Ser residues within a nuclear translocation signal (NTS), which allows binding to importin7 and a subsequent penetration via nuclear pores. However, the regulation of this process and the protein kinases involved are not yet clear. METHODS: To answer this point we developed specific anti phospho-SPS antibody, used this and other antibodies in Western blots and crystalized the phospho-mimetic mutated ERK. RESULTS: Here we show that the phosphorylation of both Ser residues is mediated mainly by casein kinase 2 (CK2) and that active ERK may assist in the phosphorylation of the N-terminal Ser. We also demonstrate that the phosphorylation is dependent on the release of ERK from cytoplasmic anchoring proteins. Crystal structure of the phosphomimetic ERK revealed that the NTS phosphorylation creates an acidic patch in ERK. Our model is that in resting cells ERK is bound to cytoplasmic anchors, which prevent its NTS phosphorylation. Upon stimulation, phosphorylation of the ERK TEY domain releases ERK and allows phosphorylation of its NTS by CK2 and active ERK to generate a negatively charged patch in ERK, binding to importin 7 and nuclear translocation. CONCLUSION: These results provide an important role of CK2 in regulating nuclear ERK activities.

Hoefavidin: A dimeric bacterial avidin with a C-terminal binding tail.

Dimeric avidins are a newly discovered subgroup of the avidin family that bind biotin with high affinity. Their dimeric configuration is a quaternary substructure of the classical tetrameric avidins which lacks the requirement of the critical Trp that defines the tetramer and dictates the tenacious interaction with biotin. Hoefavidin, derived from the bacterium Hoeflea phototrophica DFL-43(T), is the third characterized member of the dimeric avidin subfamily. Like the other members of this group, hoefavidin is a thermostable protein that contains a disulfide bridge between Cys57 and Cys88, thereby connecting and stabilizing the L3,4 and L5,6 loops. This represents a distinctive characteristic of dimeric avidins that compensates for the lack of Trp and enables their dimeric configuration. The X-ray structure of the intact hoefavidin revealed unique crystal packing generated by an octameric cylindrical structure wherein the C-termini segments of each monomer is introduced into the entrance of the biotin-binding site of an adjacent non-canonical monomer. This anomaly in the protein structure served as a lead toward the design of specific binding peptides. We screened for specific hoefavidin binding peptides derived from the C-terminal region and two peptides were obtained that bind a truncated form of hoefavidin (lacking the last 10 amino acids) with dissociation constants of 10(-5)M. The crystal structure of short hoefavidin complexed with a C-terminal derived peptide revealed the mode of binding. These peptides may form the basis of novel and reversible binders for dimeric avidins.

The p38ß mitogen-activated protein kinase possesses an intrinsic autophosphorylation activity, generated by a short region composed of the α-G helix and MAPK insert.

Protein kinases are regulated by a large number of mechanisms that vary from one kinase to another. However, a fundamental activation mechanism shared by all protein kinases is phosphorylation of a conserved activation loop threonine residue. This is achieved in many cases via autophosphorylation. The mechanism and structural basis for autophosphorylation are not clear and are in fact enigmatic because this phosphorylation occurs when the kinase is in its inactive conformation. Unlike most protein kinases, MAP kinases are not commonly activated by autophosphorylation but rather by MEK-dependent phosphorylation. Here we show that p38ß, a p38 isoform that is almost identical to p38α, is exceptional and spontaneously autoactivates by autophosphorylation. We identified a 13-residue-long region composed of part of the αG-helix and the MAPK insert that triggers the intrinsic autophosphorylation activity of p38ß. When inserted into p38α, this fragment renders it spontaneously active in vitro and in mammalian cells. We further found that an interaction between the N terminus and a particular region of the C-terminal extension suppresses the intrinsic autophosphorylation of p38ß in mammalian cells. Thus, this study identified the structural motif responsible for the unique autophosphorylation capability of p38ß and the motif inhibiting this activity in living cells. It shows that the MAPK insert and C-terminal extension, structural motifs that are unique to MAPKs, play a critical role in controlling autophosphorylation.

Characterization of Arabidopsis NEET reveals an ancient role for NEET proteins in iron metabolism.

The NEET family is a newly discovered group of proteins involved in a diverse array of biological processes, including autophagy, apoptosis, aging, diabetes, and reactive oxygen homeostasis. They form a novel structure, the NEET fold, in which two protomers intertwine to form a two-domain motif, a cap, and a unique redox-active labile 2Fe-2S cluster binding domain. To accelerate the functional study of NEET proteins, as well as to examine whether they have an evolutionarily conserved role, we identified and characterized a plant NEET protein. Here, we show that the Arabidopsis thaliana At5g51720 protein (At-NEET) displays biochemical, structural, and biophysical characteristics of a NEET protein. Phenotypic characterization of At-NEET revealed a key role for this protein in plant development, senescence, reactive oxygen homeostasis, and Fe metabolism. A role in Fe metabolism was further supported by biochemical and cell biology studies of At-NEET in plant and mammalian cells, as well as mutational analysis of its cluster binding domain. Our findings support the hypothesis that NEET proteins have an ancient role in cells associated with Fe metabolism.

DEF pocket in p38α facilitates substrate selectivity and mediates autophosphorylation.

Signaling processes are primarily promoted by molecular recognition and corresponding protein-protein interactions. One of the key eukaryotic signaling pathways is the MAP kinase cascade involved in vital cellular processes such as cell proliferation, differentiation, apoptosis, and stress response. The principle recognition site of MAP kinases, the common docking (CD) region, forms selective interactions with substrates, upstream activators, and phosphatases. A second docking site, defined as the DEF site interaction pocket (DEF pocket), is formed subsequent to ERK2 and p38α activation. Both crystal structures of p38α in its dually phosphorylated form and of intrinsically active mutants showed the DEF pocket, giving motivation for studying its role in substrate activation and selectivity. Mutating selected DEF pocket residues significantly decreased the phosphorylation levels of three p38α substrates (ATFII, Elk-1, and MBP) with no apparent effect on the phosphorylation of MK2 kinase. Conversely, mutating the CD region gave the opposite effect, suggesting p38α substrates can be classified into DEF-dependent and DEF-independent substrates. In addition, mutating DEF pocket residues decreased the autophosphorylation capability of intrinsically active p38α mutants, suggesting DEF-mediated trans-autophosphorylation in p38α. These results could contribute to understanding substrate selectivity of p38α and serve as a platform for designing p38α-selective DEF site blockers, which partially inhibit p38α binding DEF-dependent substrates, whereas maintaining its other functions intact. In this context, preliminary results using synthetic peptides reveal significant inhibition of substrate phosphorylation by activated p38α.

A point mutation in the [2Fe-2S] cluster binding region of the NAF-1 protein (H114C) dramatically hinders the cluster donor properties.

NAF-1 is an important [2Fe-2S] NEET protein associated with human health and disease. A mis-splicing mutation in NAF-1 results in Wolfram Syndrome type 2, a lethal childhood disease. Upregulation of NAF-1 is found in epithelial breast cancer cells, and suppression of NAF-1 expression by knockdown significantly suppresses tumor growth. Key to NAF-1 function is the NEET fold with its [2Fe-2S] cluster. In this work, the high-resolution structure of native NAF-1 was determined to 1.65 Šresolution (R factor = 13.5%) together with that of a mutant in which the single His ligand of its [2Fe-2S] cluster, His114, was replaced by Cys. The NAF-1 H114C mutant structure was determined to 1.58 Šresolution (R factor = 16.0%). All structural differences were localized to the cluster binding site. Compared with native NAF-1, the [2Fe-2S] clusters of the H114C mutant were found to (i) be 25-fold more stable, (ii) have a redox potential that is 300 mV more negative and (iii) have their cluster donation/transfer function abolished. Because no global structural differences were found between the mutant and the native (wild-type) NAF-1 proteins, yet significant functional differences exist between them, the NAF-1 H114C mutant is an excellent tool to decipher the underlying biological importance of the [2Fe-2S] cluster of NAF-1 in vivo.

Allostery in the ferredoxin protein motif does not involve a conformational switch.

Regulation of protein function via cracking, or local unfolding and refolding of substructures, is becoming a widely recognized mechanism of functional control. Oftentimes, cracking events are localized to secondary and tertiary structure interactions between domains that control the optimal position for catalysis and/or the formation of protein complexes. Small changes in free energy associated with ligand binding, phosphorylation, etc., can tip the balance and provide a regulatory functional switch. However, understanding the factors controlling function in single-domain proteins is still a significant challenge to structural biologists. We investigated the functional landscape of a single-domain plant-type ferredoxin protein and the effect of a distal loop on the electron-transfer center. We find the global stability and structure are minimally perturbed with mutation, whereas the functional properties are altered. Specifically, truncating the L1,2 loop does not lead to large-scale changes in the structure, determined via X-ray crystallography. Further, the overall thermal stability of the protein is only marginally perturbed by the mutation. However, even though the mutation is distal to the iron-sulfur cluster (∼20 Å), it leads to a significant change in the redox potential of the iron-sulfur cluster (57 mV). Structure-based all-atom simulations indicate correlated dynamical changes between the surface-exposed loop and the iron-sulfur cluster-binding region. Our results suggest intrinsic communication channels within the ferredoxin fold, composed of many short-range interactions, lead to the propagation of long-range signals. Accordingly, protein interface interactions that involve L1,2 could potentially signal functional changes in distal regions, similar to what is observed in other allosteric systems.

Structural adaptation of a thermostable biotin-binding protein in a psychrophilic environment.

Shwanavidin is an avidin-like protein from the marine proteobactrium Shewanella denitrificans, which exhibits an innate dimeric structure while maintaining high affinity toward biotin. A unique residue (Phe-43) from the L3,4 loop and a distinctive disulfide bridge were shown to account for the high affinity toward biotin. Phe-43 emulates the function and position of the critical intermonomeric Trp that characterizes the tetrameric avidins but is lacking in shwanavidin. The 18 copies of the apo-monomer revealed distinctive snapshots of L3,4 and Phe-43, providing rare insight into loop flexibility, binding site accessibility, and psychrophilic adaptation. Nevertheless, as in all avidins, shwanavidin also displays high thermostability properties. The unique features of shwanavidin may provide a platform for the design of a long sought after monovalent form of avidin, which would be ideal for novel types of biotechnological application.

Self-assembly of a dimeric avidin into unique higher-order oligomers.

The dimeric avidin family has been expanded in recent years to include many new members. All of them lack the intermonomeric Trp that plays a critical role in biotin-binding. Nevertheless, these new members of the avidins maintain the high affinity towards biotin. Additionally, all of the dimeric avidins share a very unique property: namely, the cylindrical oligomerization in the crystal structure. The newest member described here, agroavidin from the agrobacterium, Rhizobium sp. AAP43, shares their important structural features. However, the affinity of agroavidin towards biotin is lower than all other members of the avidin family, due to the presence of phenylalanine instead of a conserved tyrosine in the biotin-binding site. Mutating this phenylalanine into tyrosine regenerated the high affinity, which emphasizes the importance of this particular tyrosine residue. Another unique feature that distinguishes agroavidin from the other dimeric avidins is that it does not produce oligomers in its crystal structure. In order to understand the factors that promote oligomerization in dimeric avidins, we exchanged the C-terminal region of agroavidin with that of hoefavidin that produced octamers. This exchange resulted in a decamer rather than an octamer. This unusual outcome demonstrates the impact of the C-terminal region on the ability to produce oligomers. The decameric assembly of agroavidin expands the avidin-biotin toolbox even further and could well pave the path into new biotin-based technologies. Moreover, uncovering the factors that induce dimeric avidins into oligomeric assemblies may aid in better understanding the general molecular determinants that promote oligomerization.

Wilavidin - a novel member of the avidin family that forms unique biotin-binding hexamers.

Nature's optimization of protein functions is a highly intricate evolutionary process. In addition to optimal tertiary folding, the intramolecular recognition among the monomers that generate higher-order quaternary arrangements is driven by stabilizing interactions that have a pivotal role for ideal activity. Homotetrameric avidin and streptavidin are regularly utilized in many applications, whereby their ultra-high affinity toward biotin is dependent on their quaternary arrangements. In recent years, a new subfamily of avidins was discovered that comprises homodimers rather than tetramers, in which the high affinity toward biotin is maintained. Intriguingly, several of the respective dimers have been shown to assemble into higher-order cylindrical hexamers or octamers that dissociate into dimers upon biotin binding. Here, we present wilavidin, a newly discovered member of the dimeric subfamily, forming hexamers in the apo form, which are uniquely maintained upon biotin binding with six high-affinity binding sites. Removal of the short C-terminal segment of wilavidin resulted in the presence of the dimer only, thus emphasizing the role of this segment in stabilizing the hexamer. Utilization of a hexavalent biotin-binding form of avidin would be beneficial for expanding the biotechnological toolbox. Additionally, this unique family of dimeric avidins and their propensity to oligomerize to hexamers or octamers can serve as a basis for protein oligomerization and intermonomeric recognition as well as cumulative interactions that determine molecular assemblies.

An anti-diabetic drug targets NEET (CISD) proteins through destabilization of their [2Fe-2S] clusters.

Elevated levels of mitochondrial iron and reactive oxygen species (ROS) accompany the progression of diabetes, negatively impacting insulin production and secretion from pancreatic cells. In search for a tool to reduce mitochondrial iron and ROS levels, we arrived at a molecule that destabilizes the [2Fe-2S] clusters of NEET proteins (M1). Treatment of db/db diabetic mice with M1 improved hyperglycemia, without the weight gain observed with alternative treatments such as rosiglitazone. The molecular interactions of M1 with the NEET proteins mNT and NAF-1 were determined by X-crystallography. The possibility of controlling diabetes by molecules that destabilize the [2Fe-2S] clusters of NEET proteins, thereby reducing iron-mediated oxidative stress, opens a new route for managing metabolic aberration such as in diabetes.

Chimeric avidin shows stability against harsh chemical conditions--biochemical analysis and 3D structure.

Avidin and its bacterial analog streptavidin have been widely used in applications in life sciences. Recently, we described a highly thermostable engineered avidin, called chimeric avidin, which is a hybrid of avidin and avidin-related protein 4. Here, we report a protocol for pilot-scale production in E. coli and the X-ray structure of chimeric avidin. The ligand-binding properties of chimeric avidin were explored with isothermal titration calorimetry. We found chimeric avidin to be more stable against various harsh organic solvents at elevated temperatures compared to avidin and streptavidin. The properties of chimeric avidin make it a potential tool for new applications in biotechnology.

Conformational bias imposed by source microseeds results in structural ambiguity.

The p38 MAP kinase pathway is an essential component of numerous cellular signalling networks which are usually activated in response to extracellular environmental stress conditions. In addition to the canonical activation, several alternative activation pathways have been identified for p38; one of these, in which p38 is initially phosphorylated on Tyr323 and consequently autoactivated, is exclusive to T cells and is induced by TCR activation. Intrinsically active and inactive mutants at position 323 have been developed in order to evaluate the structural changes that occur upon TCR-induced activation. In order to promote crystal growth, cross streak-seeding techniques were utilized. This technique has gained popularity in promoting crystal growth when spontaneous nucleation induces critical defects or is being entirely hindered. The crystal characteristics of some mutants were highly similar to those of the wild-type source seeds (form A). In contrast, other mutants crystallized spontaneously with a different space group and molecular packing (form B). One of the active mutants (Y323T) crystallized in both crystal forms, displaying different packing characteristics and significant differences in molecular conformation that were clearly dictated by the source seeds. This implies that the source seeds used in cross streak-seeding could, in some cases, impose bias on the structural outcome of the studied molecule. Such incidents could occur when the conformational freedom permits crystal packing while not reflecting the authentic structure.

Novel clostridial cell-surface hemicellulose-binding CBM3 proteins.

A novel member of the family 3 carbohydrate-binding modules (CBM3s) is encoded by a gene (Cthe_0271) in Clostridium thermocellum which is the most highly expressed gene in the bacterium during its growth on several types of biomass substrates. Surprisingly, CtCBM3-0271 binds to at least two different types of xylan, instead of the common binding of CBM3s to cellulosic substrates. CtCBM3-0271 was crystallized and its three-dimensional structure was solved and refined to a resolution of 1.8 Å. In order to learn more about the role of this type of CBM3, a comparative study with its orthologue from Clostridium clariflavum (encoded by the Clocl_1192 gene) was performed, and the three-dimensional structure of CcCBM3-1192 was determined to 1.6 Šresolution. Carbohydrate binding by CcCBM3-1192 was found to be similar to that by CtCBM3-0271; both exhibited binding to xylan rather than to cellulose. Comparative structural analysis of the two CBM3s provided a clear functional correlation of structure and binding, in which the two CBM3s lack the required number of binding residues in their cellulose-binding strips and thus lack cellulose-binding capabilities. This is an enigma, as CtCBM3-0271 was reported to be a highly expressed protein when the bacterium was grown on cellulose. An additional unexpected finding was that CcCBM3-1192 does not contain the calcium ion that was considered to play a structural stabilizing role in the CBM3 family. Despite the lack of calcium, the five residues that form the calcium-binding site are conserved. The absence of calcium results in conformational changes in two loops of the CcCBM3-1192 structure. In this context, superposition of the non-calcium-binding CcCBM3-1192 with CtCBM3-0271 and other calcium-binding CBM3s reveals a much broader two-loop region in the former compared with CtCBM3-0271.

The butyrylcholinesterase K variant confers structurally derived risks for Alzheimer pathology.

The K variant of butyrylcholinesterase (BChE-K, 20% incidence) is a long debated risk factor for Alzheimer disease (AD). The A539T substitution in BChE-K is located at the C terminus, which is essential both for BChE tetramerization and for its capacity to attenuate beta-amyloid (Abeta) fibril formation. Here, we report that BChE-K is inherently unstable as compared with the "usual" BChE (BChE-U), resulting in reduced hydrolytic activity and predicting prolonged acetylcholine maintenance and protection from AD. A synthetic peptide derived from the C terminus of BChE-K (BSP-K), which displayed impaired intermolecular interactions, was less potent in suppressing Abeta oligomerization than its BSP-U counterpart. Correspondingly, highly purified recombinant human rBChE-U monomers suppressed beta-amyloid fibril formation less effectively than dimers, which also protected cultured neuroblastoma cells from Abeta neurotoxicity. Dual activity structurally derived changes due to the A539T substitution can thus account for both neuroprotective characteristics caused by sustained acetylcholine levels and elevated AD risk due to inefficient interference with amyloidogenic processes.

Challenging semi-bootstrapping molecular-replacement strategy reveals intriguing crystal packing of rhizavidin.

The structure of rhizavidin, the first dimeric member of the avidin family which maintains high affinity towards biotin, was determined to high resolution by SeMet SAD. Consequently, the structure of the rhizavidin-biotin complex was determined by molecular-replacement methods using the apo structure as the search model; this ran into complications and required combined programs as well as bootstrapping approaches. Although present as a dimer in solution, rhizavidin packs as unique oligomers in both crystal forms. The novel insights derived from the unique molecular-replacement procedure and the crystal-driven oligomeric forms in this work may have utililty in biotechological and nanotechnological applications.