PMID- 7761440
OWN - NLM
STAT- MEDLINE
DCOM- 19950629
LR  - 20190501
IS  - 0027-8424 (Print)
IS  - 1091-6490 (Electronic)
IS  - 0027-8424 (Linking)
VI  - 92
IP  - 11
DP  - 1995 May 23
TI  - Structure-assisted redesign of a protein-zinc-binding site with femtomolar 
      affinity.
PG  - 5017-21
AB  - We have inserted a fourth protein ligand into the zinc coordination polyhedron of 
      carbonic anhydrase II (CAII) that increases metal affinity 200-fold (Kd = 20 fM). 
      The three-dimensional structures of threonine-199-->aspartate (T199D) and 
      threonine-199-->glutamate (T199E) CAIIs, determined by x-ray crystallographic 
      methods to resolutions of 2.35 Angstrum and 2.2 Angstrum, respectively, reveal a 
      tetrahedral metal-binding site consisting of H94, H96, H119, and the engineered 
      carboxylate side chain, which displaces zinc-bound hydroxide. Although the 
      stereochemistry of neither engineered carboxylate-zinc interaction is comparable 
      to that found in naturally occurring protein zinc-binding sites, protein-zinc 
      affinity is enhanced in T199E CAII demonstrating that ligand-metal separation is 
      a significant determinant of carboxylate-zinc affinity. In contrast, the 
      three-dimensional structure of threonine-199-->histidine (T199H) CAII, determined 
      to 2.25-Angstrum resolution, indicates that the engineered imidazole side chain 
      rotates away from the metal and does not coordinate to zinc; this results in a 
      weaker zinc-binding site. All three of these substitutions nearly obliterate CO2 
      hydrase activity, consistent with the role of zinc-bound hydroxide as catalytic 
      nucleophile. The engineering of an additional protein ligand represents a general 
      approach for increasing protein-metal affinity if the side chain can adopt a 
      reasonable conformation and achieve inner-sphere zinc coordination. Moreover, 
      this structure-assisted design approach may be effective in the development of 
      high-sensitivity metal ion biosensors.
FAU - Ippolito, J A
AU  - Ippolito JA
AD  - Department of Chemistry, University of Pennsylvania, Philadelphia 19104-6323, 
      USA.
FAU - Baird, T T Jr
AU  - Baird TT Jr
FAU - McGee, S A
AU  - McGee SA
FAU - Christianson, D W
AU  - Christianson DW
FAU - Fierke, C A
AU  - Fierke CA
LA  - eng
SI  - PDB/1CCS
SI  - PDB/1CCT
SI  - PDB/1CCU
GR  - GM08558/GM/NIGMS NIH HHS/United States
GR  - GM40602/GM/NIGMS NIH HHS/United States
GR  - GM45614/GM/NIGMS NIH HHS/United States
PT  - Comparative Study
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, Non-P.H.S.
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - Proc Natl Acad Sci U S A
JT  - Proceedings of the National Academy of Sciences of the United States of America
JID - 7505876
RN  - 0 (Ligands)
RN  - 0 (Recombinant Proteins)
RN  - EC 4.2.1.1 (Carbonic Anhydrases)
RN  - J41CSQ7QDS (Zinc)
SB  - IM
MH  - Binding Sites
MH  - Carbonic Anhydrases/*chemistry/*metabolism
MH  - Catalysis
MH  - Cloning, Molecular
MH  - Crystallography, X-Ray
MH  - Drug Design
MH  - Escherichia coli
MH  - Humans
MH  - Kinetics
MH  - Ligands
MH  - Models, Molecular
MH  - Molecular Sequence Data
MH  - Mutagenesis, Site-Directed
MH  - *Protein Conformation
MH  - Recombinant Proteins/chemistry/metabolism
MH  - Sensitivity and Specificity
MH  - Zinc/*metabolism
PMC - PMC41839
EDAT- 1995/05/23 00:00
MHDA- 2001/03/28 10:01
PMCR- 1995/11/23
CRDT- 1995/05/23 00:00
PHST- 1995/05/23 00:00 [pubmed]
PHST- 2001/03/28 10:01 [medline]
PHST- 1995/05/23 00:00 [entrez]
PHST- 1995/11/23 00:00 [pmc-release]
AID - 10.1073/pnas.92.11.5017 [doi]
PST - ppublish
SO  - Proc Natl Acad Sci U S A. 1995 May 23;92(11):5017-21. doi: 
      10.1073/pnas.92.11.5017.