How the biotin–streptavidin interaction was made even stronger: investigation via crystallography and a chimaeric tetramer

The interaction between SA (streptavidin) and biotin is one of the strongest non-covalent interactions in Nature. SA is a widely used tool and a paradigm for protein–ligand interactions. We previously developed a SA mutant, termed Tr (traptavidin), possessing a 10-fold lower off-rate for biotin, with increased mechanical and thermal stability. In the present study, we determined the crystal structures of apo-Tr and biotin–Tr at 1.5 Å resolution. In apo-SA the loop (L3/4), near biotin's valeryl tail, is typically disordered and open, but closes upon biotin binding. In contrast, L3/4 was shut in both apo-Tr and biotin–Tr. The reduced flexibility of L3/4 and decreased conformational change on biotin binding provide an explanation for Tr's reduced biotin off- and on-rates. L3/4 includes Ser45, which forms a hydrogen bond to biotin consistently in Tr, but erratically in SA. Reduced breakage of the biotin–Ser45 hydrogen bond in Tr is likely to inhibit the initiating event in biotin's dissociation pathway. We generated a Tr with a single biotin-binding site rather than four, which showed a simi-larly low off-rate, demonstrating that Tr's low off-rate was governed by intrasubunit effects. Understanding the structural features of this tenacious interaction may assist the design of even stronger affinity tags and inhibitors.

[1]  Randy J. Read,et al.  Dauter Iterative model building , structure refinement and density modification with the PHENIX AutoBuild wizard , 2007 .

[2]  K Dane Wittrup,et al.  Monovalent, reduced-size quantum dots for imaging receptors on living cells , 2008, Nature Methods.

[3]  H. Gaub,et al.  Intermolecular forces and energies between ligands and receptors. , 1994, Science.

[4]  M. Howarth,et al.  Targeting quantum dots to surface proteins in living cells with biotin ligase. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[5]  O. Laitinen,et al.  Genetically engineered avidins and streptavidins , 2006, Cellular and Molecular Life Sciences CMLS.

[6]  P A Kollman,et al.  Absolute and relative binding free energy calculations of the interaction of biotin and its analogs with streptavidin using molecular dynamics/free energy perturbation approaches , 1993, Proteins.

[7]  T. Lybrand,et al.  A structural snapshot of an intermediate on the streptavidin-biotin dissociation pathway. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[8]  C. Cantor,et al.  Genetic engineering of streptavidin, a versatile affinity tag. , 1998, Journal of chromatography. B, Biomedical sciences and applications.

[9]  G. Weiss,et al.  Dissecting the Streptavidin–Biotin Interaction by Phage‐Displayed Shotgun Scanning , 2002, Chembiochem : a European journal of chemical biology.

[10]  Ole N Jensen,et al.  Protein hydrogen exchange measured at single-residue resolution by electron transfer dissociation mass spectrometry. , 2009, Analytical chemistry.

[11]  M. Wilchek,et al.  Ligand Exchange between Proteins , 2002, The Journal of Biological Chemistry.

[12]  D. Cramb,et al.  A two-photon excitation fluorescence cross-correlation assay for a model ligand-receptor binding system using quantum dots. , 2006, Biophysical journal.

[13]  H. Gruber,et al.  Accurate measurement of avidin and streptavidin in crude biofluids with a new, optimized biotin-fluorescein conjugate. , 1999, Biochimica et biophysica acta.

[14]  C. Cantor,et al.  Engineered single-chain dimeric streptavidins with an unexpected strong preference for biotin-4-fluorescein. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[15]  S. Evoy,et al.  Electronic structure, binding energy, and solvation structure of the streptavidin-biotin supramolecular complex: ONIOM and 3D-RISM study. , 2009, The journal of physical chemistry. B.

[16]  Patrick Rodriguez,et al.  Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[17]  Pieter C Dorrestein,et al.  A monovalent streptavidin with a single femtomolar biotin binding site , 2006, Nature Methods.

[18]  Mark Howarth,et al.  Separating speed and ability to displace roadblocks during DNA translocation by FtsK , 2010, The EMBO journal.

[19]  D. Dressman,et al.  Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[20]  David Baker,et al.  Macromolecular modeling with rosetta. , 2008, Annual review of biochemistry.

[21]  D. M. Simons,et al.  Structure-Based Design of Synthetic Azobenzene Ligands for Streptavidin , 1994 .

[22]  J. Chatal,et al.  Antibody pretargeting advances cancer radioimmunodetection and radioimmunotherapy. , 2006, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[23]  Ethan A Merritt,et al.  Cooperative hydrogen bond interactions in the streptavidin–biotin system , 2006, Protein science : a publication of the Protein Society.

[24]  Emile G. Bruneau,et al.  Identification of Nicotinic Acetylcholine Receptor Recycling and Its Role in Maintaining Receptor Density at the Neuromuscular Junction In Vivo , 2005, The Journal of Neuroscience.

[25]  Terry P Lybrand,et al.  Dynamics of the streptavidin-biotin complex in solution and in its crystal lattice: distinct behavior revealed by molecular simulations. , 2009, The journal of physical chemistry. B.

[26]  Collaborative Computational,et al.  The CCP4 suite: programs for protein crystallography. , 1994, Acta crystallographica. Section D, Biological crystallography.

[27]  Michael K Gilson,et al.  Host-guest complexes with protein-ligand-like affinities: computational analysis and design. , 2009, Journal of the American Chemical Society.

[28]  B Tidor,et al.  Substantial energetic improvement with minimal structural perturbation in a high affinity mutant antibody. , 2004, Journal of molecular biology.

[29]  I. Kuntz,et al.  The maximal affinity of ligands. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[30]  P. Bongrand,et al.  Dissecting streptavidin-biotin interaction with a laminar flow chamber. , 2002, Biophysical journal.

[31]  M. Wilchek,et al.  Crystal structure of rhizavidin: insights into the enigmatic high-affinity interaction of an innate biotin-binding protein dimer. , 2009, Journal of molecular biology.

[32]  M. Levy,et al.  Directed evolution of streptavidin variants using in vitro compartmentalization. , 2008, Chemistry & biology.

[33]  K N Houk,et al.  The origins of femtomolar protein-ligand binding: hydrogen-bond cooperativity and desolvation energetics in the biotin-(strept)avidin binding site. , 2007, Journal of the American Chemical Society.

[34]  P. Stayton,et al.  Structural studies of the streptavidin binding loop , 1997, Protein science : a publication of the Protein Society.

[35]  M. Howarth,et al.  Electrophilic Affibodies Forming Covalent Bonds to Protein Targets* , 2009, The Journal of Biological Chemistry.

[36]  M. Wilchek,et al.  Sodium dodecyl sulfate‐polyacrylamide gel electrophoretic method for assessing the quaternary state and comparative thermostability of avidin and streptavidin , 1996, Electrophoresis.

[37]  P. Utz,et al.  HIT: a versatile proteomics platform for multianalyte phenotyping of cytokines, intracellular proteins and surface molecules , 2008, Nature Medicine.

[38]  T. Lybrand,et al.  Streptavidin-biotin binding energetics. , 1999, Biomolecular engineering.

[39]  R. Vessella,et al.  Development of new biotin/streptavidin reagents for pretargeting. , 1999, Biomolecular engineering.

[40]  P. Tavan,et al.  Ligand Binding: Molecular Mechanics Calculation of the Streptavidin-Biotin Rupture Force , 1996, Science.

[41]  Jones Ml,et al.  Noncooperativity of biotin binding to tetrameric streptavidin. , 1995 .

[42]  A. Chilkoti,et al.  Site-directed mutagenesis studies of the high-affinity streptavidin-biotin complex: contributions of tryptophan residues 79, 108, and 120. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[43]  Andrew G. Leach,et al.  Binding Affinities of Host—Guest, Protein—Ligand, and Protein—Transition-State Complexes , 2004 .

[44]  J. Wendoloski,et al.  Structural origins of high-affinity biotin binding to streptavidin. , 1989, Science.

[45]  Early mechanistic events in biotin dissociation from streptavidin , 2002, Nature Structural Biology.

[46]  Vincent B. Chen,et al.  Correspondence e-mail: , 2000 .

[47]  Vincent T. Moy,et al.  A streptavidin variant with slower biotin dissociation and increased mechanostability , 2010, Nature Methods.

[48]  B. Katz Binding of biotin to streptavidin stabilizes intersubunit salt bridges between Asp61 and His87 at low pH. , 1997, Journal of molecular biology.

[49]  J. Foote,et al.  Breaking the affinity ceiling for antibodies and T cell receptors. , 2000, Proceedings of the National Academy of Sciences of the United States of America.