I02-Macromolecular Crystallography
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Petra
Dzianová
,
Seiya
Asai
,
Martina
Chrudinová
,
Lucie
Kosinová
,
Pavlo
Potalitsyn
,
Pavel
Šácha
,
Romana
Hadravová
,
Irena
Selicharová
,
Jan
Kříž
,
Johan P.
Turkenburg
,
Andrzej Marek
Brzozowski
,
Jiří
Jiráček
,
Lenka
Žáková
Diamond Proposal Number(s):
[13587]
Open Access
Abstract: Insulin is produced and stored inside the pancreatic β-cell secretory granules, where it is assumed to form Zn2+-stabilized oligomers. However, the actual storage forms of this hormone and the impact of zinc ions on insulin production in vivo are not known. Our initial X-ray fluorescence experiment on granules from native Langerhans islets and insulinoma-derived INS-1E cells revealed a considerable difference in the zinc content. This led our further investigation to evaluate the impact of the intra-granular Zn2+ levels on the production and storage of insulin in different model β-cells. Here, we systematically compared zinc and insulin contents in the permanent INS-1E and BRIN-BD11 β-cells and in the native rat pancreatic islets by flow cytometry, confocal microscopy, immunoblotting, specific messenger RNA (mRNA) and total insulin analysis. These studies revealed an impaired insulin production in the permanent β-cell lines with the diminished intracellular zinc content. The drop in insulin and Zn2+ levels was paralleled by a lower expression of ZnT8 zinc transporter mRNA and hampered proinsulin processing/folding in both permanent cell lines. To summarize, we showed that the disruption of zinc homeostasis in the model β-cells correlated with their impaired insulin and ZnT8 production. This indicates a need for in-depth fundamental research about the role of zinc in insulin production and storage.
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Oct 2020
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Nikolaj Kulahin
Roed
,
Cristina M.
Viola
,
Ole
Kristensen
,
Gerd
Schluckebier
,
Mathias
Norrman
,
Waseem
Sajid
,
John D.
Wade
,
Asser Sloth
Andersen
,
Claus
Kristensen
,
Timothy R.
Ganderton
,
Johan P.
Turkenburg
,
Pierre
De Meyts
,
Andrzej M.
Brzozowski
Diamond Proposal Number(s):
[1221, 7864]
Open Access
Abstract: The insulin/insulin-like growth factor signalling axis is an evolutionary ancient and highly conserved hormonal system involved in the regulation of metabolism, growth and lifespan in animals. Human insulin is stored in the pancreas, while insulin-like growth factor-1 (IGF-1) is maintained in blood in complexes with IGF-binding proteins (IGFBP1–6). Insect insulin-like polypeptide binding proteins (IBPs) have been considered as IGFBP-like structural and functional homologues. Here, we report structures of the Drosophila IBP Imp-L2 in its free form and bound to Drosophila insulin-like peptide 5 and human IGF-1. Imp-L2 contains two immunoglobulin-like fold domains and its architecture is unrelated to human IGFBPs, suggesting a distinct strategy for bioavailability regulation of insulin-like hormones. Similar hormone binding modes may exist in other insect vectors, as the IBP sequences are highly conserved. Therefore, these findings may open research routes towards a rational interference of transmission of diseases such as malaria, dengue and yellow fevers.
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Sep 2018
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Vladimír
Palivec
,
Cristina M.
Viola
,
Mateusz
Kozak
,
Timothy R.
Ganderton
,
Květoslava
Křížková
,
Johan P.
Turkenburg
,
Petra
Halušková
,
Lenka
Žáková
,
Jiří
Jiráček
,
Pavel
Jungwirth
,
Andrzej M.
Brzozowski
Diamond Proposal Number(s):
[7864, 9948]
Open Access
Abstract: Human insulin is a pivotal protein hormone controlling metabolism, growth and ageing, and whose malfunctioning underlies diabetes, some cancers and neuro-degeneration. Despite its central position in human physiology, the in vivo oligomeric state and conformation of insulin in its storage granules in the pancreas are not known. In contrast, many in vitro structures of hexamers of this hormone are available, which fall into three conformational states: T6, T3Rf3 and R6. As there is strong evidence for accumulation of neurotransmitters, such as serotonin and dopamine, in insulin storage granules in pancreatic β-cells, we probed by molecular dynamics (MD) and protein crystallography (PC) if these endogenous ligands affect and stabilize insulin oligomers. Parallel studies independently converged on the observation that serotonin binds well within the insulin hexamer (site I), stabilizing it in the T3R3 conformation. Both methods indicated serotonin binding on the hexamer surface (site III) as well. MD, but not PC, indicated that dopamine was also a good site III ligand. Some of the PC studies also included arginine, which may be abundant in insulin granules upon processing of pro-insulin, and stable T3R3 hexamers loaded with both serotonin and arginine were obtained. The MD and PC results were supported further by in solution spectroscopic studies with R-state specific chromophore. Our results indicate that the T3R3 oligomer is a plausible insulin pancreatic storage form, resulting from its complex interplay with neurotransmitters, and pro-insulin processing products. These findings may have implications for clinical insulin formulations.
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Mar 2017
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I03-Macromolecular Crystallography
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Jitka
Viková
,
Michaela
Collinsová
,
Emília
Kletvíková
,
Miloš
Buděšínský
,
Vojtěch
Kaplan
,
Lenka
Žáková
,
Václav
Veverka
,
Rozálie
Hexnerová
,
Roberto J. Tarazona
Aviñó
,
Jana
Straková
,
Irena
Selicharová
,
Václav
Vaněk
,
Daniel W
Wright
,
Christopher J
Watson
,
Johan P
Turkenburg
,
Andrzej M
Brzozowski
,
Jiří
Jiráček
Diamond Proposal Number(s):
[7864]
Open Access
Abstract: Insulin is a key hormone of human metabolism with major therapeutic importance for both types of diabetes. New insulin analogues with more physiological profiles and better glycemic control are needed, especially analogues that preferentially bind to the metabolic B-isoform of insulin receptor (IR-B). Here, we aimed to stabilize and modulate the receptor-compatible conformation of insulin by covalent intra-chain crosslinking within its B22–B30 segment, using the CuI-catalyzed Huisgen 1,3-dipolar cycloaddition reaction of azides and alkynes. This approach resulted in 14 new, systematically crosslinked insulin analogues whose structures and functions were extensively characterized and correlated. One of the analogues, containing a B26–B29 triazole bridge, was highly active in binding to both IR isoforms, with a significant preference for IR-B. Our results demonstrate the potential of chemistry-driven modulation of insulin function, also shedding new light on the functional importance of hormone’s B-chain C-terminus for its IR-B specificity.
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Jan 2016
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[9948]
Abstract: Heparan sulfate (HS) is a glycosaminoglycan that forms a key component of the extracellular matrix (ECM). Breakdown of HS is carried out by heparanase (HPSE), an endo-β-glucuronidase of the glycoside hydrolase 79 (GH79) family. Overexpression of HPSE results in breakdown of extracellular HS and release of stored growth factors and hence is strongly linked to cancer metastasis. Here we present crystal structures of human HPSE at 1.6-Å to 1.9-Å resolution that reveal how an endo-acting binding cleft is exposed by proteolytic activation of latent proHPSE. We used oligosaccharide complexes to map the substrate-binding and sulfate-recognition motifs. These data shed light on the structure and interactions of a key enzyme involved in ECM maintenance and provide a starting point for the design of HPSE inhibitors for use as biochemical tools and anticancer therapeutics.
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Nov 2015
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I02-Macromolecular Crystallography
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Diamond Proposal Number(s):
[7864]
Open Access
Abstract: The structural characterization of the insulin-insulin receptor (IR) interaction still lacks the conformation of the crucial B21-B30 insulin region, which must be different from that in its storage forms to ensure effective receptor binding. Here, it is shown that insulin analogues modified by natural amino acids at the TyrB26 site can represent an active form of this hormone. In particular, [AsnB26]-insulin and [GlyB26]-insulin attain a B26-turn-like conformation that differs from that in all known structures of the native hormone. It also matches the receptor interface, avoiding substantial steric clashes. This indicates that insulin may attain a B26-turn-like conformation upon IR binding. Moreover, there is an unexpected, but significant, binding specificity of the AsnB26 mutant for predominantly the metabolic B isoform of the receptor. As it is correlated with the B26 bend of the B-chain of the hormone, the structures of AsnB26 analogues may provide the first structural insight into the structural origins of differential insulin signalling through insulin receptor A and B isoforms.
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Oct 2014
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[9948]
Open Access
Abstract: The N-terminus of the B-chain of insulin may adopt two alternative conformations designated as the T- and R-states. Despite the recent structural insight into insulin–insulin receptor (IR) complexes, the physiological relevance of the T/R transition is still unclear. Hence, this study focused on the rational design, synthesis, and characterization of human insulin analogues structurally locked in expected R- or T-states. Sites B3, B5, and B8, capable of affecting the conformation of the N-terminus of the B-chain, were subjects of rational substitutions with amino acids with specific allowed and disallowed dihedral φ and ψ main-chain angles. α-Aminoisobutyric acid was systematically incorporated into positions B3, B5, and B8 for stabilization of the R-state, and N-methylalanine and d-proline amino acids were introduced at position B8 for stabilization of the T-state. IR affinities of the analogues were compared and correlated with their T/R transition ability and analyzed against their crystal and nuclear magnetic resonance structures. Our data revealed that (i) the T-like state is indeed important for the folding efficiency of (pro)insulin, (ii) the R-state is most probably incompatible with an active form of insulin, (iii) the R-state cannot be induced or stabilized by a single substitution at a specific site, and (iv) the B1–B8 segment is capable of folding into a variety of low-affinity T-like states. Therefore, we conclude that the active conformation of the N-terminus of the B-chain must be different from the “classical” T-state and that a substantial flexibility of the B1–B8 segment, where GlyB8 plays a key role, is a crucial prerequisite for an efficient insulin–IR interaction.
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Jun 2014
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I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: Despite the recent first structural insight into the insulin-insulin receptor complex, the role of the C terminus of the B-chain of insulin in this assembly remains unresolved. Previous studies have suggested that this part of insulin must rearrange to reveal amino acids crucial for interaction with the receptor. The role of the invariant PheB24, one of the key residues of the hormone, in this process remains unclear. For example, the B24 site functionally tolerates substitutions to d-amino acids but not to l-amino acids. Here, we prepared and characterized a series of B24-modified insulin analogues, also determining the structures of [d-HisB24]-insulin and [HisB24]-insulin. The inactive [HisB24]-insulin molecule is remarkably rigid due to a tight accommodation of the l-His side chain in the B24 binding pocket that results in the stronger tethering of B25-B28 residues to the protein core. In contrast, the highly active [d-HisB24]-insulin is more flexible, and the reverse chirality of the B24C? atom swayed the d-HisB24 side chain into the solvent. Furthermore, the pocket vacated by PheB24 is filled by PheB25, which mimics the PheB24 side and main chains. The B25?B24 downshift results in a subsequent downshift of TyrB26 into the B25 site and the departure of B26-B30 residues away from the insulin core. Our data indicate the importance of the aromatic l-amino acid at the B24 site and the structural invariance/integrity of this position for an effective binding of insulin to its receptor. Moreover, they also suggest limited, B25-B30 only, unfolding of the C terminus of the B-chain upon insulin activation.
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Feb 2013
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I24-Microfocus Macromolecular Crystallography
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John
Menting
,
Jonathan
Whittaker
,
Mai B.
Margetts
,
Linda J.
Whittaker
,
Geoffrey K.-W.
Kong
,
Brian J.
Smith
,
Christopher J.
Watson
,
Lenka
záková
,
Emília
Kletvíková
,
Jiří
Jiráček
,
Shu Jin
Chan
,
Donald F.
Steiner
,
Guy G.
Dodson
,
Andrzej M.
Brzozowski
,
Michael A.
Weiss
,
Colin W.
Ward
,
Michael C.
Lawrence
Abstract: Insulin receptor signalling has a central role in mammalian biology, regulating cellular metabolism, growth, division, differentiation and survival. Insulin resistance contributes to the pathogenesis of type 2 diabetes mellitus and the onset of Alzheimer’s disease; aberrant signalling occurs in diverse cancers, exacerbated by cross-talk with the homologous type 1 insulin-like growth factor receptor (IGF1R). Despite more than three decades of investigation, the three-dimensional structure of the insulin–insulin receptor complex has proved elusive, confounded by the complexity of producing the receptor protein. Here we present the first view, to our knowledge, of the interaction of insulin with its primary binding site on the insulin receptor, on the basis of four crystal structures of insulin bound to truncated insulin receptor constructs. The direct interaction of insulin with the first leucine-rich-repeat domain (L1) of insulin receptor is seen to be sparse, the hormone instead engaging the insulin receptor carboxy-terminal α-chain (αCT) segment, which is itself remodelled on the face of L1 upon insulin binding. Contact between insulin and L1 is restricted to insulin B-chain residues. The αCT segment displaces the B-chain C-terminal β-strand away from the hormone core, revealing the mechanism of a long-proposed conformational switch in insulin upon receptor engagement. This mode of hormone–receptor recognition is novel within the broader family of receptor tyrosine kinases. We support these findings by photo-crosslinking data that place the suggested interactions into the context of the holoreceptor and by isothermal titration calorimetry data that dissect the hormone–insulin receptor interface. Together, our findings provide an explanation for a wealth of biochemical data from the insulin receptor and IGF1R systems relevant to the design of therapeutic insulin analogues.
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Jan 2013
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Abstract: Design of inhibitors for N-myristoyltransferase (NMT), an enzyme responsible for protein trafficking in Plasmodium falciparum, the most lethal species of parasites that cause malaria, is described. Chemistry-driven optimization of compound 1 from a focused NMT inhibitor library led to the identification of two early lead compounds 4 and 25, which showed good enzyme and cellular potency and excellent selectivity over human NMT. These molecules provide a valuable starting point for further development.
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Oct 2012
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