PSI Structural Biology Knowledgebase

PSI | Structural Biology Knowledgebase
Header Icons
E-Collection

Related Articles
Families in Gene Neighborhoods
June 2015
Expanding the Reach of SAD
April 2015
Greasing the Path for SFX
January 2015
Time-Resolved Crystallography with HATRX
December 2014
Structures Without Damage
August 2014
Error Prevention
July 2014
A Refined Refinement Strategy
May 2014
Membrane Proteome: Microcrystals Yield Big Data
April 2014
Optimizing Damage
February 2014
Getting Better at Low Resolution
January 2014
Building a Structural Library
November 2013
Drug Discovery: Identifying Dynamic Networks by CONTACT
October 2013
Microbiome: Solid-State NMR, Crystallized
September 2013
Fluorescence- and Chromatography-Based Protein Thermostability Assay
October 2012
Insert Here
October 2012
Native phasing
August 2012
Smaller may be better
April 2012
Metal mates
February 2012
Not so cool
December 2011
One from many
August 2011
Rosetta hone
July 2011
Solutions in the solution
June 2011
Beyond crystals, solutions, and powders
May 2011
Snapshot crystallography
March 2011
FERM-ly bound
February 2011
A new amphiphile for crystallizing membrane proteins
January 2011
'Super-resolution' large complexes
December 2010
Proteinase K and Digalacturonic Acid
September 2010
Some crystals like it hot
May 2010
Tips for crystallizing membrane proteins in lipidic mesophases
February 2010
Tackling the phase problem
November 2009
Crystallizing glycoproteins
September 2009
Crystals from recalcitrant proteins
August 2009
Tips for crystallizing membrane proteins
June 2009
Chaperone-assisted crystallography
March 2009
An “X-ray” ruler
January 2009
Methylation boosts protein crystallization
December 2008

Technology Topics Crystallography

Metal mates

SBKB [doi:10.1038/sbkb.2011.63]
Technical Highlight - February 2012
Short description: Experimental validation of a designed metal-mediated dimer highlights the potential of computational methods.

Comparison of the MID1 model (green) and crystal structure (blue). Spheres indicate zinc ions. Figure courtesy of Brian Kuhlman.

The diversity of protein-protein interactions presents a significant challenge to the rational design of novel interfaces. While directed evolution is a powerful method, computational design allows specification of new interfaces in desired locations and orientations. This approach does, however, have drawbacks associated with the enormous conformational search space and requires accurate modeling of atomic details at the interface.

Kuhlman and colleagues (PSI NESG) have used known coordination spheres of metals to design and experimentally validate the structure of a metal-mediated symmetric homodimer. The authors used RosettaMatch to select 42,000 two-residue zinc binding matches on 600 known monomeric protein scaffolds. The matches were used to combinatorially generate 500,000 dimeric coordinate sets with no backbone clashes and acceptable tetrahedral coordination geometry for the zinc ions. These starting structures were input into a design step that iteratively optimized both backbone geometry and sequence while minimizing the number of mutations. Finally, the models were evaluated by Rosetta analysis of the binding interfaces and zinc positions, allowing the authors to select eight designs for experimental validation.

Although seven of the eight designs expressed poorly or formed higher-order oligomers, metal interface design 1 (MID1) dimerized as expected. The authors used fluorescence polarization to measure an impressive 200-fold increase in binding affinity upon zinc coordination. Although conformational heterogeneity precluded the use of NMR, the authors were able to determine the crystal structures of wild-type and mutant forms of MID1. Structures of the MID1-apo dimer do not resemble the computational design. However, the authors observed a reorientation of the dimer interface in the zinc-coordinated structure in which the zinc atoms and interface residues are very close to the designed positions in a nearly symmetric dimer. The structure further shows that the zinc ions are coordinated by three histidines compared to the four designed. The authors repaired the four-residue zinc coordination by mutation or cobalt substitution while noting that four-histidine coordination is a very rare natural occurrence. The results contribute to our understanding of protein complexes and have practical implications for therapeutic protein design and synthetic biology.

Michael A. Durney

References

  1. B. S. Der et al. Metal-mediated affinity and orientation specificity in a computationally designed protein homodimer.
    J. Am. Chem. Soc. (17 November 2011). doi:10.1021/ja208015j

Structural Biology Knowledgebase ISSN: 1758-1338
Funded by a grant from the National Institute of General Medical Sciences of the National Institutes of Health