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PDBsum entry 2p49

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protein ligands Protein-protein interface(s) links
Hydrolase/immune system PDB id
2p49
Jmol
Contents
Protein chains
119 a.a. *
123 a.a. *
Ligands
PO4
Waters ×350
* Residue conservation analysis
PDB id:
2p49
Name: Hydrolase/immune system
Title: Complex of a camelid single-domain vhh antibody fragment wit at 1.4a resolution: native mono_1 crystal form
Structure: Ribonuclease pancreatic. Chain: a. Synonym: rnase 1, rnase a. Antibody cab-rn05. Chain: b. Fragment: truncated at residue 121. Engineered: yes
Source: Bos taurus. Cattle. Organism_taxid: 9913. Camelus dromedarius. Arabian camel. Organism_taxid: 9838. Expressed in: escherichia coli. Expression_system_taxid: 562
Resolution:
1.38Å     R-factor:   0.157     R-free:   0.185
Authors: V.Tereshko,S.Uysal,K.Margalef,A.Koide,A.A.Kossiakoff,S.Koide
Key ref:
A.Koide et al. (2007). Exploring the capacity of minimalist protein interfaces: interface energetics and affinity maturation to picomolar KD of a single-domain antibody with a flat paratope. J Mol Biol, 373, 941-953. PubMed id: 17888451 DOI: 10.1016/j.jmb.2007.08.027
Date:
11-Mar-07     Release date:   28-Aug-07    
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chain
Pfam   ArchSchema ?
P61823  (RNAS1_BOVIN) -  Ribonuclease pancreatic
Seq:
Struc:
150 a.a.
119 a.a.
Protein chain
Pfam   ArchSchema ?
A2KD57  (A2KD57_LAMGL) -  Hi113 protein (Fragment)
Seq:
Struc:
132 a.a.
123 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 30 residue positions (black crosses)

 Enzyme reactions 
   Enzyme class: Chain A: E.C.3.1.27.5  - Pancreatic ribonuclease.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: Endonucleolytic cleavage to nucleoside 3'-phosphates and 3'-phosphooligonucleotides ending in C-P or U-P with 2',3'-cyclic phosphate intermediates.
 Gene Ontology (GO) functional annotation 
  GO annot!
  Cellular component     extracellular region   1 term 
  Biological process     metabolic process   3 terms 
  Biochemical function     nucleic acid binding     6 terms  

 

 
DOI no: 10.1016/j.jmb.2007.08.027 J Mol Biol 373:941-953 (2007)
PubMed id: 17888451  
 
 
Exploring the capacity of minimalist protein interfaces: interface energetics and affinity maturation to picomolar KD of a single-domain antibody with a flat paratope.
A.Koide, V.Tereshko, S.Uysal, K.Margalef, A.A.Kossiakoff, S.Koide.
 
  ABSTRACT  
 
A major architectural class in engineered binding proteins ("antibody mimics") involves the presentation of recognition loops off a single-domain scaffold. This class of binding proteins, both natural and synthetic, has a strong tendency to bind a preformed cleft using a convex binding interface (paratope). To explore their capacity to produce high-affinity interfaces with diverse shape and topography, we examined the interface energetics and explored the affinity limit achievable with a flat paratope. We chose a minimalist paratope limited to two loops found in a natural camelid heavy-chain antibody (VHH) that binds to ribonuclease A. Ala scanning of the VHH revealed only three "hot spot" side chains and additional four residues important for supporting backbone-mediated interactions. The small number of critical residues suggested that this is not an optimized paratope. Using selection from synthetic combinatorial libraries, we enhanced its affinity by >100-fold, resulting in variants with Kd as low as 180 pM with no detectable loss of binding specificity. High-resolution crystal structures revealed that the mutations induced only subtle structural changes but extended the network of interactions. This resulted in an expanded hot spot region including four additional residues located at the periphery of the paratope with a concomitant loss of the so-called "O-ring" arrangement of energetically inert residues. These results suggest that this class of simple, single-domain scaffolds is capable of generating high-performance binding interfaces with diverse shape. More generally, they suggest that highly functional interfaces can be designed without closely mimicking natural interfaces.
 
  Selected figure(s)  
 
Figure 3.
Fig. 3. High-resolution X-ray crystal structures of wild-type and affinity-matured V[H]Hs in complex with RNaseA. (a) A comparison of the two structures after superposition of the RNaseA portion. The wild type is shown in gray and the mutant in green. (b) CDR1 and CDR3 residues of the wild-type (gray) and mutant (green) V[H]Hs. The dashed line divides CDR1 and CDR3. Mutations in the affinity-matured V[H]H are shown in orange and labeled. (c) The epitope on RNaseA in the wild-type (gray) and mutant (green) complexes. Y76 has two conformers in the mutant structure.
Figure 4.
Fig. 4. Energetic contributions of the paratope residues of the affinity-matured V[H]H probed with Ala-scanning mutagenesis. (a) Effects of Ala substitution on antigen binding expressed in terms of ΔΔG (left axis) and the ratio of K[d] (K[d]^wild type/K[d]^mutant; right axis). Positions 95, 96, 98, 100–100b and 100d were not tested because (i) Gly residue is critical (labeled with “G”), (ii) no sequence convergence was found in library screening (labeled with “X”) or (iii) the Tyr is involved in scaffolding (labeled with “Y”). The value for L99, a noncontacting residue, is shown in gray. (b) The surface representation of the affinity-matured V[H]H. The black line encloses residues within 5 Å of antigen atoms. Red and cyan surfaces are for residues where Ala substitution causes > 100-fold and 10- to 100-fold increase in K[d], respectively. Gray surfaces indicate critical residues that were not tested with Ala scanning. The remaining CDR1 and CDR3 residues are shown in light blue and dark blue, respectively. The four residues that expand the hot spot (W29, D97, R100c and R102) are labeled. (c) CDR1 and CDR3 residues are shown as sticks and the V[H]H scaffold as a cartoon model.
 
  The above figures are reprinted from an Open Access publication published by Elsevier: J Mol Biol (2007, 373, 941-953) copyright 2007.  
  Figures were selected by the author.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
20445236 Z.S.Derewenda (2010).
Application of protein engineering to enhance crystallizability and improve crystal properties.
  Acta Crystallogr D Biol Crystallogr, 66, 604-615.  
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
19576999 L.Bloom, and V.Calabro (2009).
FN3: a new protein scaffold reaches the clinic.
  Drug Discov Today, 14, 949-955.  
  19298050 S.Koide, and S.S.Sidhu (2009).
The importance of being tyrosine: lessons in molecular recognition from minimalist synthetic binding proteins.
  ACS Chem Biol, 4, 325-334.  
19477632 S.Koide (2009).
Engineering of recombinant crystallization chaperones.
  Curr Opin Struct Biol, 19, 449-457.  
18602117 R.N.Gilbreth, K.Esaki, A.Koide, S.S.Sidhu, and S.Koide (2008).
A dominant conformational role for amino acid diversity in minimalist protein-protein interfaces.
  J Mol Biol, 381, 407-418.
PDB codes: 3csb 3csg
18445622 V.Tereshko, S.Uysal, A.Koide, K.Margalef, S.Koide, and A.A.Kossiakoff (2008).
Toward chaperone-assisted crystallography: protein engineering enhancement of crystal packing and X-ray phasing capabilities of a camelid single-domain antibody (VHH) scaffold.
  Protein Sci, 17, 1175-1187.
PDB codes: 2p42 2p43 2p44 2p45 2p46 2p47 2p48
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.