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- PDB-3kq6: Enhancing the Therapeutic Properties of a Protein by a Designed Z... -

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Basic information

Entry
Database: PDB / ID: 3kq6
TitleEnhancing the Therapeutic Properties of a Protein by a Designed Zinc-Binding Site, Structural principles of a novel long-acting insulin analog
Components
  • Insulin A chain
  • Insulin B chain
KeywordsHORMONE / zinc-binding site / long-acting insulin analog / receptor binding / Carbohydrate metabolism / Cleavage on pair of basic residues / Diabetes mellitus / Disease mutation / Disulfide bond / Glucose metabolism / Pharmaceutical / Secreted
Function / homology
Function and homology information


negative regulation of NAD(P)H oxidase activity / negative regulation of glycogen catabolic process / regulation of cellular amino acid metabolic process / Signaling by Insulin receptor / IRS activation / nitric oxide-cGMP-mediated signaling / negative regulation of fatty acid metabolic process / Insulin processing / negative regulation of feeding behavior / regulation of protein secretion ...negative regulation of NAD(P)H oxidase activity / negative regulation of glycogen catabolic process / regulation of cellular amino acid metabolic process / Signaling by Insulin receptor / IRS activation / nitric oxide-cGMP-mediated signaling / negative regulation of fatty acid metabolic process / Insulin processing / negative regulation of feeding behavior / regulation of protein secretion / positive regulation of peptide hormone secretion / Regulation of gene expression in beta cells / positive regulation of respiratory burst / positive regulation of dendritic spine maintenance / alpha-beta T cell activation / negative regulation of acute inflammatory response / negative regulation of respiratory burst involved in inflammatory response / negative regulation of protein secretion / fatty acid homeostasis / Synthesis, secretion, and deacylation of Ghrelin / positive regulation of glycogen biosynthetic process / positive regulation of lipid biosynthetic process / Signal attenuation / FOXO-mediated transcription of oxidative stress, metabolic and neuronal genes / negative regulation of gluconeogenesis / positive regulation of nitric oxide mediated signal transduction / regulation of protein localization to plasma membrane / COPI-mediated anterograde transport / negative regulation of lipid catabolic process / negative regulation of oxidative stress-induced intrinsic apoptotic signaling pathway / positive regulation of insulin receptor signaling pathway / negative regulation of reactive oxygen species biosynthetic process / transport vesicle / positive regulation of protein autophosphorylation / Insulin receptor recycling / insulin-like growth factor receptor binding / NPAS4 regulates expression of target genes / positive regulation of protein metabolic process / neuron projection maintenance / endoplasmic reticulum-Golgi intermediate compartment membrane / positive regulation of brown fat cell differentiation / activation of protein kinase B activity / positive regulation of glycolytic process / Insulin receptor signalling cascade / positive regulation of mitotic nuclear division / Regulation of insulin secretion / positive regulation of nitric-oxide synthase activity / positive regulation of long-term synaptic potentiation / endosome lumen / positive regulation of cytokine production / acute-phase response / positive regulation of protein secretion / regulation of transmembrane transporter activity / positive regulation of cell differentiation / positive regulation of glucose import / negative regulation of proteolysis / regulation of synaptic plasticity / wound healing / insulin receptor binding / negative regulation of protein catabolic process / positive regulation of neuron projection development / hormone activity / cognition / Golgi lumen / vasodilation / positive regulation of protein localization to nucleus / glucose metabolic process / regulation of protein localization / glucose homeostasis / cell-cell signaling / insulin receptor signaling pathway / positive regulation of NF-kappaB transcription factor activity / PI5P, PP2A and IER3 Regulate PI3K/AKT Signaling / positive regulation of cell growth / secretory granule lumen / protease binding / positive regulation of MAPK cascade / positive regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction / positive regulation of cell migration / G protein-coupled receptor signaling pathway / Amyloid fiber formation / endoplasmic reticulum lumen / Golgi membrane / negative regulation of gene expression / positive regulation of cell population proliferation / positive regulation of gene expression / regulation of DNA-templated transcription / extracellular space / extracellular region / identical protein binding
Similarity search - Function
Insulin / Insulin family / Insulin/IGF/Relaxin family / Insulin, conserved site / Insulin family signature. / Insulin-like / Insulin / insulin-like growth factor / relaxin family. / Insulin-like superfamily
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.9 Å
AuthorsWan, Z.L. / Hu, S.Q. / Whittaker, L. / Phillips, N.B. / Whittake, J. / Ismail-Beigi, F. / Weiss, M.A.
Citation
Journal: J.Biol.Chem. / Year: 2010
Title: Supramolecular protein engineering: design of zinc-stapled insulin hexamers as a long acting depot.
Authors: Phillips, N.B. / Wan, Z.L. / Whittaker, L. / Hu, S.Q. / Huang, K. / Hua, Q.X. / Whittaker, J. / Ismail-Beigi, F. / Weiss, M.A.
#1: Journal: Biochemistry / Year: 2004
Title: Enhancing the activity of insulin at the receptor interface: crystal structure and photo-cross-linking of A8 analogues.
Authors: Wan, Z.L. / Xu, B. / Chu, Y.C. / Li, B. / Nakagawa, S.H. / Qu, Y. / Hu, S.Q. / Katsoyannis, P.G. / Weiss, M.A.
#2: Journal: Philos.Trans.R.Soc.London,Ser.B / Year: 1988
Title: The structure of 2zn pig insulin crystal at 1.5 A resolution
Authors: Baker, E.N. / Blundell, T.L. / Cutfield, J.F. / Cutfield, S.M. / Dodson, E.J. / Dodson, G.G. / Hodgkin, D. / Isaacs, N.W. / Reynolds, C.D.
#3: Journal: Nature / Year: 1976
Title: Structure of insulin in 4-zinc insulin
Authors: Bentley, G. / Dodson, E. / Dodson, G. / Hodgkin, D. / Mercola, D.
#4: Journal: Nature / Year: 1989
Title: Phenol stabilizes more helix in a new symmetrical zinc insulin hexamer
Authors: Derewenda, U. / Derewenda, Z. / Dodson, E. / Dodson, G. / Reynold, C. / Smith, G. / Sparks, C. / Swenson, D.
History
DepositionNov 17, 2009Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 23, 2010Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2Oct 13, 2021Group: Database references / Derived calculations
Category: database_2 / struct_conn ...database_2 / struct_conn / struct_ref_seq_dif / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Revision 1.3Sep 6, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Insulin A chain
B: Insulin B chain
C: Insulin A chain
D: Insulin B chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)12,08910
Polymers11,7274
Non-polymers3616
Water1,47782
1
A: Insulin A chain
B: Insulin B chain
C: Insulin A chain
D: Insulin B chain
hetero molecules

A: Insulin A chain
B: Insulin B chain
C: Insulin A chain
D: Insulin B chain
hetero molecules

A: Insulin A chain
B: Insulin B chain
C: Insulin A chain
D: Insulin B chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)36,26630
Polymers35,18212
Non-polymers1,08418
Water21612
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_655-y+1,x-y,z1
crystal symmetry operation3_665-x+y+1,-x+1,z1
Buried area19420 Å2
ΔGint-383 kcal/mol
Surface area13160 Å2
MethodPISA
Unit cell
Length a, b, c (Å)78.091, 78.091, 36.399
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number146
Space group name H-MH3
Components on special symmetry positions
IDModelComponents
11B-101-

ZN

21D-201-

ZN

31D-202-

CL

41D-33-

HOH

51D-34-

HOH

61D-35-

HOH

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Components

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Protein/peptide , 2 types, 4 molecules ACBD

#1: Protein/peptide Insulin A chain


Mass: 2429.774 Da / Num. of mol.: 2 / Mutation: E4H, T4H / Source method: obtained synthetically / Details: biosynthetic peptide / Source: (synth.) Homo sapiens (human) / References: UniProt: P01308
#2: Protein/peptide Insulin B chain


Mass: 3433.953 Da / Num. of mol.: 2 / Source method: obtained synthetically / Details: biosynthetic peptide / Source: (synth.) Homo sapiens (human) / References: UniProt: P01308

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Non-polymers , 4 types, 88 molecules

#3: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Zn
#4: Chemical ChemComp-CL / CHLORIDE ION / Chloride


Mass: 35.453 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Cl
#5: Chemical ChemComp-IPH / PHENOL / Phenol


Mass: 94.111 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H6O
#6: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 82 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 1.82 Å3/Da / Density % sol: 32.46 %
Crystal growTemperature: 298 K / Method: vapor diffusion / pH: 8
Details: 0.02 M Tris, 0.05 M sodium citrate, 5% acetone, 0.03% phenol, 0.01% zinc acetate, pH 8.0, VAPOR DIFFUSION, temperature 298K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 4.2.2 / Wavelength: 1.0000,1.2398
DetectorType: NOIR-1 / Detector: CCD / Date: Nov 4, 2005 / Details: Double crystal
RadiationMonochromator: Double crystal / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength
IDWavelength (Å)Relative weight
111
21.23981
ReflectionResolution: 1.9→32.05 Å / Num. all: 6520 / Num. obs: 6488 / % possible obs: 99.5 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 5.49 % / Biso Wilson estimate: 27.7 Å2 / Rmerge(I) obs: 0.057 / Net I/σ(I): 14.1
Reflection shellResolution: 1.9→1.97 Å / Redundancy: 5.43 % / Rmerge(I) obs: 0.422 / Mean I/σ(I) obs: 3 / Num. unique all: 658 / % possible all: 100

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Processing

Software
NameVersionClassification
ADSCQuantumdata collection
CNSrefinement
d*TREKdata reduction
d*TREKdata scaling
CNSphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entry 1RWE

1rwe


Resolution: 1.9→32.05 Å / Isotropic thermal model: restrained / Cross valid method: THROUGHOUT / σ(F): 0 / σ(I): 0 / Stereochemistry target values: Engh & Huber
RfactorNum. reflection% reflectionSelection details
Rfree0.257 708 -Random
Rwork0.199 ---
all-6520 --
obs-6475 99.2 %-
Displacement parametersBiso mean: 43.3 Å2
Baniso -1Baniso -2Baniso -3
1--2.84 Å20 Å20 Å2
2---28.4 Å20 Å2
3---5.68 Å2
Refine analyzeLuzzati coordinate error obs: 0.27 Å / Luzzati d res low obs: 5 Å / Luzzati sigma a obs: 0.23 Å
Refinement stepCycle: LAST / Resolution: 1.9→32.05 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms818 0 12 82 912
Refine LS restraints
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONc_angle_d0.008
X-RAY DIFFRACTIONc_angle_deg1.2
X-RAY DIFFRACTIONc_dihedral_angle_d19.5
X-RAY DIFFRACTIONc_improper_angle_d0.73
LS refinement shellResolution: 1.9→2.02 Å / Rfactor Rfree error: 0.035
RfactorNum. reflection% reflection
Rfree0.34 124 -
Rwork0.269 --
obs-955 99.5 %

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