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Botulinum neurotoxins (BoNTs) are one of the most toxic proteins known to humans. Their molecular structure is comprised of three essential domains—a cell binding domain (HC), translocation domain and catalytic domain (light chain) . The HC domain facilitates the highly specific binding of BoNTs to the neuronal membrane via a dual‐receptor complex involving a protein receptor and a ganglioside. Variation in activity/toxicity across subtypes of serotype A has been attributed to changes in protein and ganglioside interactions, and their implications are important in the design of novel BoNT‐based therapeutics. Here, we present the structure of BoNT/A3 cell binding domain (HC/A3) in complex with the ganglioside GD1a at 1.75 Å resolution. The structure revealed that six residues interact with the three outermost monosaccharides of GD1a through several key hydrogen bonding interactions. A detailed comparison of structures of HC/A3 with HC/A1 revealed subtle conformational differences at the ganglioside binding site upon carbohydrate binding., The majority of botulinum neurotoxin (BoNT) serotypes require recognition of both the protein and the ganglioside receptor to gain entry in to the cell. Here, we show that the cell binding domain of BoNT A3 binds three monosaccharides of GD1a receptor. Six residues of the protein interact with this receptor through several key hydrogen bonding interactions.

The structural and functional diversity of proteins can be enhanced by numerous post-translational modifications. C-mannosylation is a rare form of glycosylation consisting of a single alpha or beta D-mannopyranose forming a carbon–carbon bond with the pyrrole ring of a tryptophan residue. Despite first being discovered in 1994, C-mannosylation is still poorly understood and 3D structures are available for only a fraction of the total predicted C-mannosylated proteins. Here, we present the first comprehensive review of C-mannosylated protein structures by analysing the data for all 10 proteins with C-mannosylation/s deposited in the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB). We analysed in detail the WXXW/WXXWXXW consensus motif and the highly conserved pair of arginine residues in thrombospondin type 1 repeat C-mannosylation sites or homologous arginine residues in other domains. Furthermore, we identified a conserved PXP sequence C-terminal of the C-mannosylation site. The PXP motif forms a tight turn region in the polypeptide chain and its universal conservation in C-mannosylated protein is worthy of further experimental study. The stabilization of C-mannopyranosyl groups was demonstrated through hydrogen bonding with arginine and other charged or polar amino acids. Where possible, the structural findings were linked to other functional studies demonstrating the role of C-mannosylation in protein stability, secretion or function. With the current technological advances in structural biology, we hope to see more progress in the study of C-mannosylation that may correspond to discoveries of novel C-mannosylation pathways and functions with implications for human health and biotechnology.

Neprilysin (NEP) is a promiscuous zinc metalloprotease with broad substrate specificity and cleaves a remarkable diversity of substrates through endopeptidase action. Two of these – amyloid-β and natriuretic peptides – implicate the enzyme in both Alzheimer’s disease and cardiovascular disease, respectively. Here, we report the creation of a catalytically inactive NEP (E584D) to determine the first peptide-bound crystal structure at 2.6 Å resolution. The structure reveals key interactions involved in substrate binding which we have identified to be conserved in other known zinc metalloproteases. In addition, the structure provides evidence for a potential exosite within the central cavity that may play a critical role in substrate positioning. Together, these results contribute to our understanding of the molecular function of NEP.

Neprilysin is a transmembrane M13 zinc metalloprotease responsible for the degradation of several biologically active peptides including insulin, enkephalin, substance P, bradykinin, endothelin-1, neurotensin and amyloid-β. The protein has received attention for its role in modulating blood pressure responses with its inhibition producing an antihypertensive response. To date, several inhibitor bound crystal structures of the human neprilysin extracellular domain have been determined, but, a structure free of bound inhibitor or substrate has yet to be reported. Here, we report the first crystal structure free of substrate or inhibitor for the extracellular catalytic domain of human neprilysin at 1.9 A resolution. This structure will provide a reference point for comparisons to future inhibitor or substrate bound structures. The neprilysin structure also reveals that a closed protein conformation can be adopted in protein crystals absent of bound substrate or inhibitor.

Botulinum neurotoxins (BoNTs) are categorised into immunologically distinct serotypes BoNT/A to /G). Each serotype can also be further divided into subtypes based on differences in amino acid sequence. BoNTs are ~150 kDa proteins comprised of three major functional domains: an N-terminal zinc metalloprotease light chain (LC), a translocation domain (HN), and a binding domain (HC). The HC is responsible for targeting the BoNT to the neuronal cell membrane, and each serotype has evolved to bind via different mechanisms to different target receptors. Most structural characterisations to date have focussed on the first identified subtype within each serotype (e.g., BoNT/A1). Subtype differences within BoNT serotypes can affect intoxication, displaying different botulism symptoms in vivo, and less emphasis has been placed on investigating these variants. This review outlines the receptors for each BoNT serotype and describes the basis for the highly specific targeting of neuronal cell membranes. Understanding receptor binding is of vital importance, not only for the generation of novel therapeutics but also for understanding how best to protect from intoxication.

Bacterial pathogens use several strategies to infect host cells, one of which involves blocking host defenses. During infection, the bacterial effector proteins GtgA, GogA, PipA, and NleC are injected into host cells by the type III secretion system (T3SS), where they suppress the proinflammatory NF-κB signaling pathway to dampen immune responses. The authors demonstrate that these effectors bind NF-κB via their DNA-mimicking regions and uncover differences in effector sequences and structures explaining the individual specificities of these effectors for distinct NF-κB subunits.

C9orf72 is associated with frontotemporal dementia (FTD) and Amyotrophic Lateral Sclerosis (ALS), both of which are devastating neurodegenerative diseases. Findings suggest that an expanded hexanucleotide repeat in the non-coding region of the C9orf72 gene is the most common cause of familial FTD and ALS. Despite considerable efforts being made towards discerning the possible disease-causing mechanism/s of this repeat expansion mutation, the biological function of C9orf72 remains unclear. Here, we present the first comprehensive genomic study on C9orf72 gene. Analysis of the genomic level organization of C9orf72 across select species revealed architectural similarity of syntenic regions between human and mouse but a lack of conservation of the repeat-harboring intron 1 sequence. Information generated in this study provides a broad genomic perspective of C9orf72 which would form a basis for subsequent experimental approaches and facilitate future mechanistic and functional studies on this gene.

Angiotensin converting enzyme (ACE) is a zinc‐dependent dipeptidyl carboxypeptidase with an essential role in blood pressure homeostasis in mammals. ACE has long been targeted in the treatment of hypertension through ACE inhibitors, however current inhibitors are known to cause severe side effects. Therefore, there is a requirement for a new generation of ACE inhibitors and structural information will be invaluable in their development. ACE is a challenging enzyme to work with due to its extensive glycosylation. As such, theDrosophila melanogaster ACE homologue, AnCE, which shares ∼60% sequence similarity with human ACE, can be used as a model for studying inhibitor binding. The presence of ligands originating from the crystallisation condition at the AnCE active site has proved an obstacle to studying the binding of new inhibitor precursors. Here we present the crystal structure of AnCE (in a new crystal form) at 1.85 Å resolution, using crystals grown under different conditions. This new structure may be more suitable for studying the binding of new compounds, with the potential of developing a new generation of improved ACE inhibitors.

Summary The prevalence of serum antibodies against Clostridium difficile (CD) toxins A and B in healthy populations have prompted interest in evaluating the therapeutic activity of intravenous immunoglobulin (IVIg) in individuals experiencing severe or recurrent C. difficile infection (CDI). Despite some promising case reports, a definitive clinical role for IVIg in CDI remains unclear. Contradictory results may be attributed to a lack of consensus regarding optimal dose, timing of administration and patient selection as well as variability in specific antibody content between commercial preparations. The purpose of this study was to investigate retrospectively the efficacy of three commercial preparations of IVIg for treating severe or recurrent CDI. In subsequent mechanistic studies using protein microarray and toxin neutralization assays, all IVIg preparations were analysed for specific binding and neutralizing antibodies (NAb) to CD antigens in vitro and the presence of anti-toxin NAbs in vivo following IVIg infusion. A therapeutic response to IVIg was observed in 41% (10 of 17) of the CDI patients. Significant variability in multi-isotype specific antibodies to a 7-plex panel of CD antigens and toxin neutralization efficacies were observed between IVIg preparations and also in patient sera before and after IVIg administration. These results extend our current understanding of population immunity to CD and support the inclusion of surface layer proteins and binary toxin antigens in CD vaccines. Future strategies could enhance IVIg treatment response rates by using protein microarray to preselect donor plasma/serum with the highest levels of anti-CD antibodies and/or anti-toxin neutralizing capacities prior to fractionation.

The control of mosquitoes is threatened by the appearance of insecticide resistance and therefore new control chemicals are urgently required. Here we show that inhibitors of mosquito peptidyl dipeptidase, a peptidase related to mammalian angiotensin-converting enzyme (ACE), are insecticidal to larvae of the mosquitoes, Aedes aegypti and Anopheles gambiae. ACE inhibitors (captopril, fosinopril and fosinoprilat) and two peptides (trypsin-modulating oostatic factor/TMOF and a bradykinin-potentiating peptide, BPP-12b) were all inhibitors of the larval ACE activity of both mosquitoes. Two inhibitors, captopril and fosinopril (a pro-drug ester of fosinoprilat), were tested for larvicidal activity. Within 24 h captopril had killed >90% of the early instars of both species with 3rd instars showing greater resistance. Mortality was also high within 24 h of exposure of 1st, 2nd and 3rd instars of An. gambiae to fosinopril. Fosinopril was also toxic to Ae. aegypti larvae, although the 1st instars appeared to be less susceptible to this pro-drug even after 72 h exposure. Homology models of the larval An. gambiae ACE proteins (AnoACE2 and AnoACE3) reveal structural differences compared to human ACE, suggesting that structure-based drug design offers a fruitful approach to the development of selective inhibitors of mosquito ACE enzymes as novel larvicides.

Mutations in Angiogenin (ANG), a member of the Ribonuclease A superfamily (also known as RNase 5) are known to be associated with Amyotrophic Lateral Sclerosis (ALS, motor neurone disease) (sporadic and familial) and Parkinson's Disease (PD). In our previous studies we have shown that ANG is expressed in neurons during neuro-ectodermal differentiation, and that it has both neurotrophic and neuroprotective functions. In addition, in an extensive study on selective ANG-ALS variants we correlated the structural changes to the effects on neuronal survival and the ability to induce stress granules in neuronal cell lines. Furthermore, we have established that ANG-ALS variants which affect the structure of the catalytic site and either decrease or increase the RNase activity affect neuronal survival. Neuronal cell lines expressing the ANG-ALS variants also lack the ability to form stress granules. Here, we report a detailed experimental structural study on eleven new ANG-PD/ALS variants which will have implications in understanding the molecular basis underlying their role in PD and ALS.

Human ACE (angiotensin-I-converting enzyme) has long been regarded as an excellent target for the treatment of hypertension and related cardiovascular diseases. Highly potent inhibitors have been developed and are extensively used in the clinic. To develop inhibitors with higher therapeutic efficacy and reduced side effects, recent efforts have been directed towards the discovery of compounds able to simultaneously block more than one zinc metallopeptidase (apart from ACE) involved in blood pressure regulation in humans, such as neprilysin and ECE-1 (endothelin-converting enzyme-1). In the present paper, we show the first structures of testis ACE [C-ACE, which is identical with the C-domain of somatic ACE and the dominant domain responsible for blood pressure regulation, at 1.97Å (1 Å=0.1 nm)] and the N-domain of somatic ACE (N-ACE, at 2.15Å) in complex with a highly potent and selective dual ACE/ECE-1 inhibitor. The structural determinants revealed unique features of the binding of two molecules of the dual inhibitor in the active site of C-ACE. In both structures, the first molecule is positioned in the obligatory binding site and has a bulky bicyclic P1′ residue with the unusual R configuration which, surprisingly, is accommodated by the large S2′ pocket. In the C-ACE complex, the isoxazole phenyl group of the second molecule makes strong pi–pi stacking interactions with the amino benzoyl group of the first molecule locking them in a ‘hand-shake’ conformation. These features, for the first time, highlight the unusual architecture and flexibility of the active site of C-ACE, which could be further utilized for structure-based design of new C-ACE or vasopeptidase inhibitors.

Angiotensin-I-converting enzyme (ACE) plays a critical role in the regulation of blood pressure through its central role in the renin-angiotensin and kallikrein-kinin systems. ACE contains two domains, the N and C domains, both of which are heavily glycosylated. Structural studies of ACE have been fraught with severe difficulties because of surface glycosylation of the protein. In order to investigate the role of glycosylation in the N domain and to create suitable forms for crystallization, we have investigated the importance of the 10 potential N-linked glycan sites using enzymatic deglycosylation, limited proteolysis, and mass spectrometry. A number of glycosylation mutants were generated via site-directed mutagenesis, expressed in CHO cells, and analyzed for enzymatic activity and thermal stability. At least eight of 10 of the potential glycan sites are glycosylated; three C-terminal sites were sufficient for expression of active N domain, whereas two N-terminal sites are important for its thermal stability. The minimally glycosylated Ndom389 construct was highly suitable for crystallization studies. The structure in the presence of an N domain-selective phosphinic inhibitor RXP407 was determined to 2.0 A resolution. The Ndom389 structure revealed a hinge region that may contribute to the breathing motion proposed for substrate binding.

The formation of blood vessels (angiogenesis) is a highly orchestrated sequence of events involving crucial receptor-ligand interactions. Angiogenesis is critical for physiological processes such as development, wound healing, reproduction, tissue regeneration, and remodeling. It also plays a major role in sustaining tumor progression and chronic inflammation. Vascular endothelial growth factor (VEGF)-B, a member of the VEGF family of angiogenic growth factors, effects blood vessel formation by binding to a tyrosine kinase receptor, VEGFR-1. There is growing evidence of the important role played by VEGF-B in physiological and pathological vasculogenesis. Development of VEGF-B antagonists, which inhibit the interaction of this molecule with its cognate receptor, would be important for the treatment of pathologies associated specifically with this growth factor. In this study, we present the crystal structure of the complex of VEGF-B with domain 2 of VEGFR-1 at 2.7 Å resolution. Our analysis reveals that each molecule of the ligand engages two receptor molecules using two symmetrical binding sites. Based on these interactions, we identify the receptor-binding determinants on VEGF-B and shed light on the differences in specificity towards VEGFR-1 among the different VEGF homologs.

The widespread and functionally varied members of the ribonuclease A (RNase A) superfamily provide an excellent opportunity to study evolutionary forces at work on a conserved protein scaffold. Representatives from the zebrafish are of particular interest as the evolutionary distance from non-ichthyic homologues is large. We conducted an exhaustive survey of available zebrafish DNA sequences and found significant polymorphism among its four known homologues. In an extension of previous nomenclature, the variants have been named RNases ZF-1a–c,-2a–d,-3a–e and-4. We present the first X-ray crystal structures of zebrafish ribonucleases, RNases ZF-1a and-3e at 1.35-and 1.85 Å resolution, respectively. Structure-based clustering with ten other ribonuclease structures indicates greatest similarity to mammalian angiogenins and amphibian ribonucleases, and supports the view that all present-day ribonucleases evolved from a progenitor with three disulphide bonds. In their details, the two structures are intriguing melting-pots of features present in ribonucleases from other vertebrate classes. Whereas in RNase ZF-1a the active site is obstructed by the C-terminal segment (as observed in angiogenin), in RNase ZF-3e the same region is open (as observed in more catalytically efficient homologues). The progenitor of present-day ribonucleases is more likely to have had an obstructive C terminus, and the relatively high similarity (late divergence) of RNases ZF-1 and-3 infers that the active site unblocking event has happened independently in different vertebrate lineages.

Angiogenesis: What We Know and Do not Know

Key Points Angiotensin-converting enzyme (ACE) is a chloride-dependent metalloenzyme that catalyses the hydrolytic cleavage of dipeptides from the carboxyl terminus of many regulatory oligopeptides. ACE is central to the renin–angiotensin system that regulates blood pressure, fluid homeostasis, and renal and vascular function. It is therefore a major target for cardiovascular therapies.ACE inhibitors (for example, captopril, enalaprilat and lisinopril) have been on the market for more than 20 years. Side effects of treatment with ACE inhibitors include cough and angioedema.ACE comprises an N- and a C-domain, each containing an active site with distinct substrates and activation properties. The design of domain-selective inhibitors might produce new drugs with improved safety and efficacy — this endeavour will be facilitated by the recent determination of the three-dimensional structure of ACE.The C-domain seems to be primarily responsible for the regulation of blood pressure. Data indicate that C-domain-selective inhibitors will have less severe side effects than current-generation inhibitors, which generally target both the N- and C-domains.In contrast to the C-domain, the N-domain seems to have relatively low affinity for the peptides that control blood pressure. It preferentially hydrolyses at least three other physiologically important peptides, so targeted inhibition of the N-domain might have novel therapeutic applications., Current-generation angiotensin-converting enzyme (ACE) inhibitors are widely used for cardiovascular diseases, including high blood pressure, heart failure, heart attack and kidney failure, and have combined annual sales in excess of US $6 billion. However, the use of these ACE inhibitors, which were developed in the late 1970s and early 1980s, is hampered by common side effects. Moreover, we now know that ACE actually consists of two parts (called the N- and C-domains) that have different functions. Therefore, the design of specific domain-selective ACE inhibitors is expected to produce next-generation drugs that might be safer and more effective. Here we discuss the structural features of current inhibitors and outline how next-generation ACE inhibitors could be designed by using the three-dimensional molecular structure of human testis ACE. The ACE structure provides a unique opportunity for rational drug design, based on a combination of in silico modelling using existing inhibitors as scaffolds and iterative lead optimization to drive the synthetic chemistry.

Glycosyltransferases (GTs) control the synthesis and structures of glycans. Inactivation and intense allelic variation in members of the GT6 family generate species-specific and individual variations in carbohydrate structures, including histo-blood group oligosaccharides, resulting in anti-glycan antibodies that target glycan-decorated pathogens. GT6 genes are ubiquitous in vertebrates but are otherwise rare, existing in a few bacteria, one protozoan, and cyanophages, suggesting lateral gene transfer. Prokaryotic GT6 genes correspond to one exon of vertebrate genes, yet their translated protein sequences are strikingly similar. Bacterial and phage GT6 genes influence the surface chemistry of bacteria, affecting their interactions, including those with vertebrate hosts.

ADP-ribosylation is one of the favored modes of cell intoxication employed by several bacteria. Clostridium difficile is recognized to be an important nosocomial pathogen associated with considerable morbidity and attributable mortality. Along with its two well known toxins, Toxin A and Toxin B, it produces an ADP-ribosylating toxin that targets monomeric actin of the target cell. Like other Clostridial actin ADP-ribosylating toxins, this binary toxin, known as C. difficile toxin (CDT), is composed of two subunits, CDTa and CDTb. In this study, we present high resolution crystal structures of CDTa in its native form (at pH 4.0, 8.5, and 9.0) and in complex with ADP-ribose donors, NAD and NADPH (at pH 9.0). The crystal structures of the native protein show "pronounced conformational flexibility" confined to the active site region of the protein and "enhanced" disorder at low pH, whereas the complex structures highlight significant differences in "ligand specificity" compared with the enzymatic subunit of a close homologue, Clostridium perfringens iota toxin. Specifically in CDTa, two of the suggested catalytically important residues (Glu-385 and Glu-387) seem to play no role or a less important role in ligand binding. These structural data provide the first detailed information on protein-donor substrate complex stabilization in CDTa, which may have implications in understanding CDT recognition.

International audience; Angiotensin-converting enzyme (ACE) is a metallopeptidase that converts angiotensin I into angiotensin II. ACE is crucial in the control of cardiovascular and renal homeostasis and fertility in mammals. In vertebrates, both transmembrane and soluble ACE, containing one or two active sites, have been characterized. So far, only soluble, single domain ACEs from invertebrates have been cloned, and these have been implicated in reproduction in insects. Furthermore, an ACE-related carboxypeptidase was recently characterized in Leishmania, a unicellular eukaryote, suggesting the existence of ACE in more distant organisms. Interestingly, in silico databank analysis revealed that bacterial DNA sequences could encode putative ACE-like proteins, strikingly similar to vertebrates' enzymes. To gain more insight into the bacterial enzymes, we cloned the putative ACE from the phytopathogenic bacterium, Xanthomonas axonopodis pv. citri, named XcACE. The 2 kb open reading frame encodes a 672-amino-acid soluble protein containing a single active site. In vitro expression and biochemical characterization revealed that XcACE is a functional 72 kDa dipeptidyl-carboxypeptidase. As in mammals, this metalloprotease hydrolyses angiotensin I into angiotensin II. XcACE is sensitive to ACE inhibitors and chloride ions concentration. Variations in the active site residues, highlighted by structural modelling, can account for the different substrate selectivity and inhibition profile compared to human ACE. XcACE characterization demonstrates that ACE is an ancestral enzyme, provoking questions about its appearance and structure/activity specialisation during the course of evolution.

Botulinum neurotoxins (BoNTs) cause muscle paralysis by selectively cleaving core components of the vesicular fusion machinery within motoneurons. Complex gangliosides initially bind into a pocket that is conserved among the seven BoNTs and tetanus neurotoxin. Productive neurotoxin uptake also requires protein receptors. The interaction site of the protein receptor within the neurotoxin is currently unknown. We report the identification and characterization of the protein receptor binding site of BoNT/B and BoNT/G. Their protein receptors, synaptotagmins I and II, bind to a pocket at the tip of their H CC (C-terminal domain of the C-terminal fragment of the heavy chain) that corresponds to the unique second carbohydrate binding site of tetanus neurotoxin, the sialic acid binding site. Substitution of amino acids in this region impaired binding to synaptotagmins and drastically decreased toxicity at mouse phrenic nerve preparations; CD-spectroscopic analyses evidenced that the secondary structure of the mutated neurotoxins was unaltered. Deactivation of the synaptotagmin binding site by single mutations led to virtually inactive BoNT/B and BoNT/G when assayed at phrenic nerve preparations of complex-ganglioside-deficient mice. Analogously, a BoNT B mutant with deactivated ganglioside and synaptotagmin binding sites lacked appreciable activity at wild-type mouse phrenic nerve preparations. Thus, these data exclude relevant contributions of any cell surface molecule other than one ganglioside and one protein receptor to the entry process of BoNTs, which substantiates the double-receptor concept. The molecular characterization of the synaptotagmin binding site provides the basis for designing a novel class of potent binding inhibitors.

Human somatic angiotensin I-converting enzyme (sACE) is a key regulator of blood pressure and an important drug target for combating cardiovascular and renal disease. sACE comprises two homologous metallopeptidase domains, N and C, joined by an inter-domain linker. Both domains are capable of cleaving the two hemoregulatory peptides angiotensin I and bradykinin, but differ in their affinities for a range of other substrates and inhibitors. Previously we determined the structure of testis ACE (C domain); here we present the crystal structure of the N domain of sACE (both in the presence and absence of the antihypertensive drug lisinopril) in order to aid the understanding of how these two domains differ in specificity and function. In addition, the structure of most of the inter-domain linker allows us to propose relative domain positions for sACE that may contribute to the domain cooperativity. The structure now provides a platform for the design of "domain-specific" second-generation ACE inhibitors.

Crystals of the complex formed between the outer membrane protein OmpC from Escherichia coli and the eukaryotic antibacterial protein lactoferrin from Camelus dromedarius (camel) have been obtained using a detergent environment. Initial data processing suggests that the crystals belong to the hexagonal space group P6, with unit-cell parameters a = b = 116.3, c = 152.4 A, alpha = beta = 90, gamma = 120 degrees. This indicated a Matthews coefficient (VM) of 3.3 A3 Da(-1), corresponding to a possible molecular complex involving four molecules of lactoferrin and two porin trimers in the unit cell (4832 amino acids; 533.8 kDa) with 63% solvent content. A complete set of diffraction data was collected to 3 A resolution at 100 K. Structure determination by molecular replacement is in progress. Structural study of this first surface-exposed membrane-protein complex with an antibacterial protein will provide insights into the mechanism of action of OmpC as well as lactoferrin.

Mutations in angiogenin (ANG), a member of the ribonuclease A superfamily, are associated with amyotrophic lateral sclerosis (ALS; sporadic and familial) and Parkinson's disease. We have previously shown that ANG is expressed in neurons during neuro-ectodermal differentiation, and that it has both neurotrophic and neuroprotective functions. Here we report the atomic resolution structure of native ANG and 11 ANG-ALS variants. We correlate the structural changes to the effects on neuronal survival and the ability to induce stress granules in neuronal cell lines. ANG-ALS variants that affect the structure of the catalytic site and either decrease or increase the RNase activity affect neuronal survival. Neuronal cell lines expressing the ANG-ALS variants also lack the ability to form stress granules. Our structure–function studies on these ANG-ALS variants are the first to provide insights into the cellular and molecular mechanisms underlying their role in ALS., Mutations in human angiogenin are implicated in the progression of amyotrophic lateral sclerosis. Thiyagarajan and colleagues show that structural differences between angiogenin variants affect neuronal survival, and the ability to induce stress granules in neuronal cell lines.

It has been proposed that the binding of Zn2+ to α-lactalbumin switches the conformation to one akin to a state intermediate in the folding of the protein. However, the high resolution x-ray crystal structure of human α-lactalbumin-Zn2+ complex at 1.7-Å resolution (pH 7.6) does not reveal any significant change in conformation from the native state. The Zn2+ ion binds specifically in the "cleft" of α-lactalbumin (the region which forms the active site of the homologous protein lysozyme). This may suggest a possible role for Zn2+ binding in lactose synthase complex. The coordination of the Zn2+ ion involves a symmetry-related molecule in the crystal, the crystal contacts being stabilized by a SO42- ion bound at the interface between three molecules.

The three-dimensional X-ray structure of human alpha-lactalbumin, an important component of milk, has been determined at 1.7 A (0.17 nm) resolution by the method of molecular replacement, using the refined structure of baboon alpha-lactalbumin as the model structure. The two proteins are known to have more than 90% amino acid sequence identity and crystallize in the same orthorhombic space group, P2(1)2(1)2. The crystallographic refinement of the structure using the simulated annealing method, resulted in a crystallographic R-factor of 0.209 for the 11,373 observed reflections (F greater than or equal to 2 sigma (F)) between 8 and 1.7 A resolution. The model comprises 983 protein atoms, 90 solvent atoms and a bound calcium ion. In the final model, the root-mean-square deviations from ideality are 0.013 A for covalent bond distances and 2.9 degrees for bond angles. Superposition of the human and baboon alpha-lactalbumin structures yields a root-mean-square difference of 0.67 A for the 123 structurally equivalent C alpha atoms. The C terminus is flexible in the human alpha-lactalbumin molecule. The striking structural resemblance between alpha-lactalbumins and C-type lysozymes emphasizes the homologous evolutionary relationship between these two classes of proteins.