PDBsum entry 2jab

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protein Protein-protein interface(s) links
De novo protein PDB id
Protein chains
124 a.a. *
Waters ×488
* Residue conservation analysis
PDB id:
Name: De novo protein
Title: A designed ankyrin repeat protein evolved to picomolar affinity to her2
Structure: H10-2-g3. Chain: a, b, c. Engineered: yes
Source: Expressed in: escherichia coli. Expression_system_taxid: 562.
1.70Å     R-factor:   0.180     R-free:   0.212
Authors: C.Zahnd,E.Wyler,J.M.Schwenk,D.Steiner,M.C.Lawrence, N.M.Mckern,F.Pecorari,C.W.Ward,T.O.Joos,A.Pluckthun
Key ref:
C.Zahnd et al. (2007). A designed ankyrin repeat protein evolved to picomolar affinity to Her2. J Mol Biol, 369, 1015-1028. PubMed id: 17466328 DOI: 10.1016/j.jmb.2007.03.028
27-Nov-06     Release date:   08-May-07    
Go to PROCHECK summary

Protein chains
No UniProt id for this chain
Struc: 124 a.a.
Key:    Secondary structure  CATH domain


DOI no: 10.1016/j.jmb.2007.03.028 J Mol Biol 369:1015-1028 (2007)
PubMed id: 17466328  
A designed ankyrin repeat protein evolved to picomolar affinity to Her2.
C.Zahnd, E.Wyler, J.M.Schwenk, D.Steiner, M.C.Lawrence, N.M.McKern, F.Pecorari, C.W.Ward, T.O.Joos, A.Plückthun.
Designed ankyrin repeat proteins (DARPins) are a novel class of binding molecules, which can be selected to recognize specifically a wide variety of target proteins. DARPins were previously selected against human epidermal growth factor receptor 2 (Her2) with low nanomolar affinities. We describe here their affinity maturation by error-prone PCR and ribosome display yielding clones with zero to seven (average 2.5) amino acid substitutions in framework positions. The DARPin with highest affinity (90 pM) carried four mutations at framework positions, leading to a 3000-fold affinity increase compared to the consensus framework variant, mainly coming from a 500-fold increase of the on-rate. This DARPin was found to be highly sensitive in detecting Her2 in human carcinoma extracts. We have determined the crystal structure of this DARPin at 1.7 A, and found that a His to Tyr mutation at the framework position 52 alters the inter-repeat H-bonding pattern and causes a significant conformational change in the relative disposition of the repeat subdomains. These changes are thought to be the reason for the enhanced on-rate of the mutated DARPin. The DARPin not bearing the residue 52 mutation has an unusually slow on-rate, suggesting that binding occurred via conformational selection of a relatively rare state, which was stabilized by this His52Tyr mutation, increasing the on-rate again to typical values. An analysis of the structural location of the framework mutations suggests that randomization of some framework residues either by error-prone PCR or by design in a future library could increase affinities and the target binding spectrum.
  Selected figure(s)  
Figure 3.
Figure 3. Crystal structure of H10-2-G3. The overall structure is shown in ribbon representation in front view, looking towards the putative binding surface. The side-chains of residues that had been randomized in the synthetic library are drawn in red. Side-chains of framework residues that had been mutated in the course of affinity maturation are drawn in blue. Note that only some of the side-chains that have been drawn are labeled.
Figure 4.
Figure 4. A stereo diagram showing detail of the accommodation of the imidazole ring of His52 in a representative N3C structure (PDB entry 1SVX) is shown in (a). Oxygen atoms are shown in red, nitrogen atoms in blue and carbon atoms in cyan. The hydrogen bonds between the imidazole ring of His52 and the respective atoms Thr49 Ogamma1 and Tyr81 O are indicated by broken red lines, as is the inter-repeat hydrogen bond between Thr46 O and Val78 N. (b) Stereo diagram showing detail of the accommodation of the phenol ring of Tyr52 in the N2C structure presented here. Oxygen atoms are shown in red, nitrogen atoms in blue and carbon atoms in copper. The single hydrogen bond between the side-chain hydroxyl of Tyr52 and the side-chain carboxylate of Asp77 is indicated by a broken red line. The increased separation (ca 6 Å) of the respective backbone atoms at positions 46 and 78 compared to the N3C structure shown in (a) is apparent. (c) Stereo diagram showing an overlay of the backbone traces of H10-2-G3 (copper) with an N3C structure (PDB entry 1SVX, cyan) generated by superimposing the Calpha atoms of residues 12 to 74 of the two structures. The rotameric change at position 52 is indicated. The 13° relative rotation of the two C-terminal ankyrin repeats of H10-2-G3 with respect to their N3C counterparts is apparent, with the grey line showing the approximate location of the rotation axis and the sphere at residue 76 indicating the approximate point in the backbone trace at which the overlaid structures begin to diverge. The Figures were generated using Molmol and POVScript+.
  The above figures are reprinted by permission from Elsevier: J Mol Biol (2007, 369, 1015-1028) copyright 2007.  
  Figures were selected by the author.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
21405972 B.Ayoglu, A.Häggmark, M.Neiman, U.Igel, M.Uhlén, J.M.Schwenk, and P.Nilsson (2011).
Systematic antibody and antigen-based proteomic profiling with microarrays.
  Expert Rev Mol Diagn, 11, 219-234.  
21214917 M.Naimuddin, S.Kobayashi, C.Tsutsui, M.Machida, N.Nemoto, T.Sakai, and T.Kubo (2011).
Directed evolution of a three-finger neurotoxin by using cDNA display yields antagonists as well as agonists of interleukin-6 receptor signaling.
  Mol Brain, 4, 2.  
21224833 R.C.Münch, M.D.Mühlebach, T.Schaser, S.Kneissl, C.Jost, A.Plückthun, K.Cichutek, and C.J.Buchholz (2011).
DARPins: an efficient targeting domain for lentiviral vectors.
  Mol Ther, 19, 686-693.  
20130104 C.Zahnd, C.A.Sarkar, and A.Plückthun (2010).
Computational analysis of off-rate selection experiments to optimize affinity maturation by directed evolution.
  Protein Eng Des Sel, 23, 175-184.  
19674966 D.E.Gloriam, S.Orchard, D.Bertinetti, E.Björling, E.Bongcam-Rudloff, C.A.Borrebaeck, J.Bourbeillon, A.R.Bradbury, Daruvar, S.Dübel, R.Frank, T.J.Gibson, L.Gold, N.Haslam, F.W.Herberg, T.Hiltke, J.D.Hoheisel, S.Kerrien, M.Koegl, Z.Konthur, B.Korn, U.Landegren, L.Montecchi-Palazzi, S.Palcy, H.Rodriguez, S.Schweinsberg, V.Sievert, O.Stoevesandt, M.J.Taussig, M.Ueffing, M.Uhlén, S.van der Maarel, C.Wingren, P.Woollard, D.J.Sherman, and H.Hermjakob (2010).
A community standard format for the representation of protein affinity reagents.
  Mol Cell Proteomics, 9, 1.  
20150180 G.Wozniak-Knopp, S.Bartl, A.Bauer, M.Mostageer, M.Woisetschläger, B.Antes, K.Ettl, M.Kainer, G.Weberhofer, S.Wiederkum, G.Himmler, G.C.Mudde, and F.Rüker (2010).
Introducing antigen-binding sites in structural loops of immunoglobulin constant domains: Fc fragments with engineered HER2/neu-binding sites and antibody properties.
  Protein Eng Des Sel, 23, 289-297.  
20694021 J.Li, and Z.Zhu (2010).
Research and development of next generation of antibody-based therapeutics.
  Acta Pharmacol Sin, 31, 1198-1207.  
20495541 J.P.Theurillat, B.Dreier, G.Nagy-Davidescu, B.Seifert, S.Behnke, U.Zürrer-Härdi, F.Ingold, A.Plückthun, and H.Moch (2010).
Designed ankyrin repeat proteins: a novel tool for testing epidermal growth factor receptor 2 expression in breast cancer.
  Mod Pathol, 23, 1289-1297.  
20412054 M.Umetsu, T.Nakanishi, R.Asano, T.Hattori, and I.Kumagai (2010).
Protein-protein interactions and selection: generation of molecule-binding proteins on the basis of tertiary structural information.
  FEBS J, 277, 2006-2014.  
  20061813 A.Beck, S.Hanala, and J.M.Reichert (2009).
4th European Antibody Congress 2008: December 1-3, 2008, Geneva, Switzerland.
  MAbs, 1, 93.  
19415706 A.Drevelle, A.Urvoas, M.B.Hamida-Rebaï, G.Van Vooren, M.Nicaise, M.Valerio-Lepiniec, M.Desmadril, C.H.Robert, and P.Minard (2009).
Disulfide bond substitution by directed evolution in an engineered binding protein.
  Chembiochem, 10, 1349-1359.  
19591507 C.F.Cervantes, P.R.Markwick, S.C.Sue, J.A.McCammon, H.J.Dyson, and E.A.Komives (2009).
Functional dynamics of the folded ankyrin repeats of IkappaB alpha revealed by nuclear magnetic resonance.
  Biochemistry, 48, 8023-8031.  
19646997 J.Huang, K.Makabe, M.Biancalana, A.Koide, and S.Koide (2009).
Structural basis for exquisite specificity of affinity clamps, synthetic binding proteins generated through directed domain-interface evolution.
  J Mol Biol, 392, 1221-1231.
PDB code: 3ch8
19723880 J.Winkler, P.Martin-Killias, A.Plückthun, and U.Zangemeister-Wittke (2009).
EpCAM-targeted delivery of nanocomplexed siRNA to tumor cells with designed ankyrin repeat proteins.
  Mol Cancer Ther, 8, 2674-2683.  
19081931 L.Kelly, M.A.McDonough, M.L.Coleman, P.J.Ratcliffe, and C.J.Schofield (2009).
Asparagine beta-hydroxylation stabilizes the ankyrin repeat domain fold.
  Mol Biosyst, 5, 52-58.
PDB codes: 2zgd 2zgg
19501012 M.Gebauer, and A.Skerra (2009).
Engineered protein scaffolds as next-generation antibody therapeutics.
  Curr Opin Chem Biol, 13, 245-255.  
19171063 M.Kawe, U.Horn, and A.Plückthun (2009).
Facile promoter deletion in Escherichia coli in response to leaky expression of very robust and benign proteins from common expression vectors.
  Microb Cell Fact, 8, 8.  
19757488 T.G.Uil, Vrij, J.Vellinga, M.J.Rabelink, S.J.Cramer, O.Y.Chan, M.Pugnali, M.Magnusson, L.Lindholm, P.Boulanger, and R.C.Hoeben (2009).
A lentiviral vector-based adenovirus fiber-pseudotyping approach for expedited functional assessment of candidate retargeted fibers.
  J Gene Med, 11, 990.  
19574456 T.V.Pavoor, Y.K.Cho, and E.V.Shusta (2009).
Development of GFP-based biosensors possessing the binding properties of antibodies.
  Proc Natl Acad Sci U S A, 106, 11895-11900.  
18654624 A.Schweizer, P.Rusert, L.Berlinger, C.R.Ruprecht, A.Mann, S.Corthésy, S.G.Turville, M.Aravantinou, M.Fischer, M.Robbiani, P.Amstutz, and A.Trkola (2008).
CD4-specific designed ankyrin repeat proteins are novel potent HIV entry inhibitors with unique characteristics.
  PLoS Pathog, 4, e1000109.  
18243686 D.Barrick, D.U.Ferreiro, and E.A.Komives (2008).
Folding landscapes of ankyrin repeat proteins: experiments meet theory.
  Curr Opin Struct Biol, 18, 27-34.  
17963718 E.Kloss, N.Courtemanche, and D.Barrick (2008).
Repeat-protein folding: new insights into origins of cooperativity, stability, and topology.
  Arch Biochem Biophys, 469, 83-99.  
18621567 M.T.Stumpp, H.K.Binz, and P.Amstutz (2008).
DARPins: a new generation of protein therapeutics.
  Drug Discov Today, 13, 695-701.  
18632570 N.D.Werbeck, P.J.Rowling, V.R.Chellamuthu, and L.S.Itzhaki (2008).
Shifting transition states in the unfolding of a large ankyrin repeat protein.
  Proc Natl Acad Sci U S A, 105, 9982-9987.  
18481120 P.Sklenovský, P.Banás, and M.Otyepka (2008).
Two C-terminal ankyrin repeats form the minimal stable unit of the ankyrin repeat protein p18INK4c.
  J Mol Model, 14, 747-759.  
18391401 T.M.Bandeiras, R.C.Hillig, P.M.Matias, U.Eberspaecher, J.Fanghänel, M.Thomaz, S.Miranda, K.Crusius, V.Pütter, P.Amstutz, M.Gulotti-Georgieva, H.K.Binz, C.Holz, A.A.Schmitz, C.Lang, P.Donner, U.Egner, M.A.Carrondo, and B.Müller-Tiemann (2008).
Structure of wild-type Plk-1 kinase domain in complex with a selective DARPin.
  Acta Crystallogr D Biol Crystallogr, 64, 339-353.
PDB code: 2v5q
The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB code is shown on the right.