Homology modelling and <sup>1</sup>H NMR studies of human leukaemia inhibitory factor

Smith, David K.; Treutlein, Herbert; Maurer, Till; Owczarek, Catherine M.; Layton, Meredith J.; Nicola, Nicos A.; Norton, Raymond S.

doi:10.1016/0014-5793(94)00785-3

Cited by 14 publications

(7 citation statements)

References 33 publications

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“…NSD and frustration in proteins have shown to be essential to protein function [43], [44]. The conserved region for receptor binding within the cytokine family is helix A [45], [46], [47], [48], [49], [50], [51]. The late formation of helix A as well as the fraying of helix A in the reduced state of the PLBs implies that the dynamics is important to receptor binding and activity of the PLBs [12].…”

Section: Resultsmentioning

confidence: 99%

“…In contrast, leptin mainly slipknots a helical-hairpin through its C-terminal loop. The closed loop also changes the dynamics of helix A which is important for activity and receptor interaction [45], [46], [47], [48], [49], [50], [51]. The NSD reveals that oxidized leptin is more flexible than the reduced state, implying that dynamics in helix A is biologically important for leptin.…”

Section: Resultsmentioning

confidence: 99%

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Pierced Lasso Bundles Are a New Class of Knot-like Motifs

et al. 2014

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A four-helix bundle is a well-characterized motif often used as a target for designed pharmaceutical therapeutics and nutritional supplements. Recently, we discovered a new structural complexity within this motif created by a disulphide bridge in the long-chain helical bundle cytokine leptin. When oxidized, leptin contains a disulphide bridge creating a covalent-loop through which part of the polypeptide chain is threaded (as seen in knotted proteins). We explored whether other proteins contain a similar intriguing knot-like structure as in leptin and discovered 11 structurally homologous proteins in the PDB. We call this new helical family class the Pierced Lasso Bundle (PLB) and the knot-like threaded structural motif a Pierced Lasso (PL). In the current study, we use structure-based simulation to investigate the threading/folding mechanisms for all the PLBs along with three unthreaded homologs as the covalent loop (or lasso) in leptin is important in folding dynamics and activity. We find that the presence of a small covalent loop leads to a mechanism where structural elements slipknot to thread through the covalent loop. Larger loops use a piercing mechanism where the free terminal plugs through the covalent loop. Remarkably, the position of the loop as well as its size influences the native state dynamics, which can impact receptor binding and biological activity. This previously unrecognized complexity of knot-like proteins within the helical bundle family comprises a completely new class within the knot family, and the hidden complexity we unraveled in the PLBs is expected to be found in other protein structures outside the four-helix bundles. The insights gained here provide critical new elements for future investigation of this emerging class of proteins, where function and the energetic landscape can be controlled by hidden topology, and should be take into account in ab initio predictions of newly identified protein targets.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Pierced Lasso Bundles Are a New Class of Knot-like Motifs

et al. 2014

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“…The model was refined in 3 stages, using simulated annealing procedures similar to the ones described for the construction of a model for human leukemia inhibitory factor (Smith et al, 1994). However, in order to speed up the calculations, the first stages of the refinement were performed in vacuum.…”

Section: Homology Modeling Of Mil-6mentioning

confidence: 99%

Structure‐Function analysis of human IL‐6: Identification of two distinct regions that are important for receptor binding

et al. 1994

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Interleukin-6 (IL-6) is a multifunctional cytokine that plays an important role in host defense. It has been predicted that IL-6 may fold as a 4 a-helix bundle structure with up-up-down-down topology. Despite a high degree of sequence similarity (42%) the human and mouse IL-6 polypeptides display distinct species-specific activities. Although human IL-6 (hIL-6) is active in both human and mouse cell assays, mouse IL-6 (mIL-6) is not active on human cells. Previously, we demonstrated that the 5 C-terminal residues of mIL-6 are important for activity, conformation, and stability , Protein Sci 2:1472-1481). To further probe the structurefunction relationship of this cytokine, we have constructed several human/mouse IL-6 hybrid molecules. Restriction endonuclease sites were introduced and used to ligate the human and mouse sequences at junction points situated at Leu-62 (Lys-65 in mIL-6) in the putative connecting loop AB between helices A and B, at Arg-113 (Val-117 in mIL-6) at the N-terminal end of helix C, at Lys-150 (Asp-152 in mIL-6) in the connecting loop CD between helices C and D, and at Leu-178 (Thr-180 in mIL-6) in helix D. Hybrid molecules consisting of various combinations of these fragments were constructed, expressed, and purified to homogeneity. The conformational integrity of the IL-6 hybrids was assessed by far-UV CD. Analysis of their biological activity in a human bioassay (using the HepG2 cell line), a mouse bioassay (using the 7TD1 cell line), and receptor binding properties indicates that at least 2 regions of hIL-6, residues 178-184 in helix D and residues 63-113 in the region incorporating part of the putative connecting loop AB through to the beginning of helix C, are critical for efficient binding to the human IL-6 receptor. For human IL-6, it would appear that interactions between residues Ala-180, Leu-181, and Met-184 and residues in the N-terminal region may be critical for maintaining the structure of the molecule; replacement of these residues with the corresponding 3 residues in mouse IL-6 correlated with a significant loss of a-helical content and a 200-fold reduction in activity in the mouse bioassay. A homology model of mIL-6 based on the X-ray structure of human granulocyte colony-stimulating factor is presented.

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“…2 (23). Similar procedures were used to construct models of IL-6 (24) and the epidermal growth factor receptor kinase (25).…”

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confidence: 99%

“…Electrostatic Calculations-As one method to find possible dimerization sites of the G-CSF-R, we performed an analysis of the electrostatic potential generated by the CRH of the G-CSF-R. The isopotential surfaces of the model were calculated by following, in general, the method described elsewhere (23,28) using the program DelPhi (29,30) with the following parameters: a probe radius of 1.4 Å, an ion exclusion layer of 2.0 Å, a salt concentration of 0.145 M, standard van der Waals radii, partial charges from the OPLS force field (27), a solvent dielectric of 80, and a solute dielectric of 2. Histidine residues were treated as having a net charge of ϩ0.5 e.…”

mentioning

confidence: 99%

Identification of a Ligand-binding Site on the Granulocyte Colony-stimulating Factor Receptor by Molecular Modeling and Mutagenesis

Layton

Iaria

Smith

et al. 1997

Journal of Biological Chemistry

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Granulocyte colony-stimulating factor (G-CSF) initiates its effects on cells of the neutrophil lineage by inducing formation of a homodimeric receptor complex. The structure of the G-CSF receptor has not yet been determined, therefore we used molecular modeling to identify regions of the receptor that were likely to be involved in ligand binding. The G-CSF receptor sequence was aligned with all the available sequences of the gp130 and growth hormone receptor families and a model of the cytokine receptor homologous domain was constructed, based on the growth hormone receptor structure. Alanine substitution mutagenesis was performed on loops and individual residues that were predicted to bind ligand. Mutant receptors were expressed in factor-dependent Ba/F3 cells and assessed for proliferation response and ligand binding. Six residues were identified that significantly reduced receptor function, with Arg 288 in the F-G loop having the greatest effect. These residues formed a binding face on the receptor model resembling the growth hormone receptor site, which suggests that the model is reasonable. However, electrostatic analysis of the model provided further evidence that the mechanism of receptor dimerization is different from that of the growth hormone receptor. The granulocyte colony-stimulating factor receptor (G-CSF-R)1 is a member of the class 1 cytokine receptor family which is defined by the presence of a cytokine receptor homology (CRH) domain that is characterized by four conserved Cys residues and a conserved WSXWS (Trp-Ser-X-Trp-Ser, where X is any amino acid) sequence (1). The receptor is expressed predominantly on cells of the neutrophil lineage (2), hence interaction with G-CSF stimulates proliferation and differentiation of neutrophils from precursor cells (3). G-CSF is required for normal neutrophil production in vivo (4). The molecular mechanisms of receptor activation and signal transduction are at present poorly understood. The receptor is most probably homodimerized by ligand binding (5-7), resulting in activation of cytoplasmic signaling pathways including the Jak-STAT pathway and the MAP kinase pathway (reviewed by Avalos (8)).Bazan (1) proposed that the conserved CRH domain is comprised of two modules of fibronectin type III (FnIII)-like structure, each containing seven ␤-strands which form two ␤-sheets. This model has been confirmed by determination of the crystal structures of several receptors including the growth hormone receptor (GH-R) (9), prolactin receptor (10), erythropoietin receptor (EPO-R) (11), and related class 2 cytokine receptors: tissue factor (12) and interferon-␥ receptor ␣ (13). The highly conserved Cys residues and WSXWS sequence are likely to be required for maintenance of the structure of the CRH domain. In the G-CSF-R, each of the CRH domain modules (termed BN and BC) have been expressed as soluble proteins, both of which bind G-CSF with low affinity (14, 15). In both the BN and BC domains, the disulfide bonds were critical for maintaining a stably folded protein and...

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Homology modelling and ¹H NMR studies of human leukaemia inhibitory factor

Cited by 14 publications

References 33 publications

Pierced Lasso Bundles Are a New Class of Knot-like Motifs

Pierced Lasso Bundles Are a New Class of Knot-like Motifs

Structure‐Function analysis of human IL‐6: Identification of two distinct regions that are important for receptor binding

Identification of a Ligand-binding Site on the Granulocyte Colony-stimulating Factor Receptor by Molecular Modeling and Mutagenesis

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Homology modelling and 1H NMR studies of human leukaemia inhibitory factor

Cited by 14 publications

References 33 publications

Pierced Lasso Bundles Are a New Class of Knot-like Motifs

Pierced Lasso Bundles Are a New Class of Knot-like Motifs

Structure‐Function analysis of human IL‐6: Identification of two distinct regions that are important for receptor binding

Identification of a Ligand-binding Site on the Granulocyte Colony-stimulating Factor Receptor by Molecular Modeling and Mutagenesis

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Homology modelling and ¹H NMR studies of human leukaemia inhibitory factor