Rigidification of a flexible protease inhibitor variant upon binding to trypsin.


:The Tyr35-->Gly replacement in bovine pancreatic trypsin inhibitor (BPTI) has previously been shown to dramatically enhance the flexibility of the trypsin-binding region of the free inhibitor and to destabilize the interaction with the protease by about 3 kcal/mol. The effects of this replacement on the enzyme-inhibitor interaction were further studied here by X-ray crystallography and isothermal titration calorimetry (ITC). The co-crystal structure of Y35G BPTI bound to trypsin was determined using 1.65 A resolution X-ray diffraction data collected from cryopreserved crystals, and a new structure of the complex with wild-type BPTI under the same conditions was determined using 1.62 A data. These structures reveal that, in contrast to the free protein, Y35G BPTI adopts a conformation nearly identical with that of the wild-type protein, with a water-filled cavity in place of the missing Tyr side-chain. The crystallographic temperature factors for the two complexes indicate that the mutant inhibitor is nearly as rigid as the wild-type protein when bound to trypsin. Calorimetric measurements show that the change in enthalpy upon dissociation of the complex is 2.5 kcal/mol less favorable for the complex containing Y35G BPTI than for the complex with the wild-type inhibitor. Thus, the destabilization of the complex resulting from the Y35G replacement is due to a more favorable change in entropy upon dissociation. The heat capacity changes for dissociation of the mutant and wild-type complexes were very similar, suggesting that the entropic effects probably do not arise from solvation effects, but are more likely due to an increase in protein conformational entropy upon dissociation of the mutant inhibitor. These results define the biophysical role of a highly conserved core residue located outside of a protein-binding interface, demonstrating that Tyr35 has little impact on the trypsin-bound BPTI structure and acts primarily to define the structure of the free protein so as to maximize binding affinity.


J Mol Biol


Hanson WM,Domek GJ,Horvath MP,Goldenberg DP




Has Abstract


2007-02-09 00:00:00














  • Structure and mechanism of L-fucose isomerase from Escherichia coli.

    abstract::The three-dimensional structure of L-fucose isomerase from Escherichia coli has been determined by X-ray crystallography at 2.5 A resolution. This ketol isomerase converts the aldose L-fucose into the corresponding ketose L-fuculose using Mn2+ as a cofactor. Being a hexamer with 64,976 Da per subunit, L-fucose isomera...

    journal_title:Journal of molecular biology

    pub_type: 杂志文章


    authors: Seemann JE,Schulz GE

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  • Molecular evolution of multisubunit RNA polymerases: sequence analysis.

    abstract::Transcription in all cellular organisms is performed by multisubunit, DNA-dependent RNA polymerases that synthesize RNA from DNA templates. Previous sequence and structural studies have elucidated the importance of shared regions common to all multisubunit RNA polymerases. In addition, RNA polymerases contain multiple...

    journal_title:Journal of molecular biology

    pub_type: 杂志文章


    authors: Lane WJ,Darst SA

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  • ABCF ATPases Involved in Protein Synthesis, Ribosome Assembly and Antibiotic Resistance: Structural and Functional Diversification across the Tree of Life.

    abstract::Within the larger ABC superfamily of ATPases, ABCF family members eEF3 in Saccharomyces cerevisiae and EttA in Escherichia coli have been found to function as ribosomal translation factors. Several other ABCFs including biochemically characterized VgaA, LsaA and MsrE confer resistance to antibiotics that target the pe...

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    pub_type: 杂志文章


    authors: Murina V,Kasari M,Takada H,Hinnu M,Saha CK,Grimshaw JW,Seki T,Reith M,Putrinš M,Tenson T,Strahl H,Hauryliuk V,Atkinson GC

    更新日期:2019-08-23 00:00:00

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    journal_title:Journal of molecular biology

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    authors: Yamamoto K,Tokunaga M,Sutoh K,Wakabayashi T,Sekine T

    更新日期:1985-05-25 00:00:00

  • Core side-chain packing and backbone conformation in Lpp-56 coiled-coil mutants.

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    journal_title:Journal of molecular biology

    pub_type: 杂志文章


    authors: Liu J,Cao W,Lu M

    更新日期:2002-05-03 00:00:00

  • Stability of a homo-dimeric Ca(2+)-binding member of the beta gamma-crystallin superfamily: DSC measurements on spherulin 3a from Physarum polycephalum.

    abstract::Spherulin 3a (S3a) from Physarum polycephalum represents the only known single-domain member of the superfamily of beta gamma eye-lens crystallins. It shares the typical two Greek-key motif and is stabilized by dimerization and Ca(2+)-binding. The temperature and denaturant-induced unfolding of S3a in the absence and ...

    journal_title:Journal of molecular biology

    pub_type: 杂志文章


    authors: Kretschmar M,Jaenicke R

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  • Hsp70 and Hsp90 of E. coli Directly Interact for Collaboration in Protein Remodeling.

    abstract::Hsp90 is a highly conserved molecular chaperone that remodels hundreds of client proteins, many involved in the progression of cancer and other diseases. It functions with the Hsp70 chaperone and numerous cochaperones. The bacterial Hsp90 functions with an Hsp70 chaperone, DnaK, but is independent of Hsp90 cochaperone...

    journal_title:Journal of molecular biology

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    authors: Genest O,Hoskins JR,Kravats AN,Doyle SM,Wickner S

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  • Initiation of DNA replication in a eukaryotic rolling-circle replicon: identification of multiple DNA-protein complexes at the geminivirus origin.

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    authors: Castellano MM,Sanz-Burgos AP,Gutiérrez C

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  • A novel disulfide bond in the SH2 Domain of the C-terminal Src kinase controls catalytic activity.

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    journal_title:Journal of molecular biology

    pub_type: 杂志文章


    authors: Mills JE,Whitford PC,Shaffer J,Onuchic JN,Adams JA,Jennings PA

    更新日期:2007-02-02 00:00:00

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    abstract::beta-Barrel membrane proteins are found in the outer membrane of Gram-negative bacteria, mitochondria, and chloroplasts. Little is known about how residues in membrane beta-barrels interact preferentially with other residues on adjacent strands. We have developed probabilistic models to quantify propensities of residu...

    journal_title:Journal of molecular biology

    pub_type: 杂志文章


    authors: Jackups R Jr,Liang J

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  • Transthyretin Interferes with Aβ Amyloid Formation by Redirecting Oligomeric Nuclei into Non-Amyloid Aggregates.

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    authors: Nilsson L,Pamrén A,Islam T,Brännström K,Golchin SA,Pettersson N,Iakovleva I,Sandblad L,Gharibyan AL,Olofsson A

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    authors: Pires JR,Taha-Nejad F,Toepert F,Ast T,Hoffmüller U,Schneider-Mergener J,Kühne R,Macias MJ,Oschkinat H

    更新日期:2001-12-14 00:00:00

  • Effects of mutations in the polymerase domain on the polymerase, RNase H and strand transfer activities of human immunodeficiency virus type 1 reverse transcriptase.

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    journal_title:Journal of molecular biology

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    authors: Gao HQ,Boyer PL,Arnold E,Hughes SH

    更新日期:1998-04-03 00:00:00

  • Evolution of three human GPCRs for higher expression and stability.

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    journal_title:Journal of molecular biology

    pub_type: 杂志文章


    authors: Dodevski I,Plückthun A

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    journal_title:Journal of molecular biology

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    authors: Espinosa JF,Muñoz V,Gellman SH

    更新日期:2001-02-23 00:00:00

  • Functional Antagonism of Human CD40 Achieved by Targeting a Unique Species-Specific Epitope.

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    authors: Yamniuk AP,Suri A,Krystek SR,Tamura J,Ramamurthy V,Kuhn R,Carroll K,Fleener C,Ryseck R,Cheng L,An Y,Drew P,Grant S,Suchard SJ,Nadler SG,Bryson JW,Sheriff S

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    authors: Cribier S,Gulik A,Fellmann P,Vargas R,Devaux PF,Luzzati V

    更新日期:1993-01-20 00:00:00

  • Wild-type alpha 1-antitrypsin is in the canonical inhibitory conformation.

    abstract::alpha 1-Antitrypsin is the archetypal member of the serine proteinase inhibitor or serpin superfamily. Members of the family show structural homology based on a dominant A beta-sheet and a mobile reactive centre loop. Our recent crystal structure of alpha 1-antitrypsin stabilized with a point mutation showed the loop ...

    journal_title:Journal of molecular biology

    pub_type: 杂志文章,评审


    authors: Elliott PR,Abrahams JP,Lomas DA

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  • Crystal structures of cobalamin-independent methionine synthase (MetE) from Streptococcus mutans: a dynamic zinc-inversion model.

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    authors: Fu TM,Almqvist J,Liang YH,Li L,Huang Y,Su XD

    更新日期:2011-09-30 00:00:00

  • The Role of Hexon Protein as a Molecular Mold in Patterning the Protein IX Organization in Human Adenoviruses.

    abstract::Adenoviruses are respiratory, ocular and enteric pathogens that form complex capsids, which are assembled from seven different structural proteins and composed of several core proteins that closely interact with the packaged dsDNA genome. The recent near-atomic resolution structures revealed that the interlacing conti...

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  • Crystal structure of the catalytic subunit of Cdc25B required for G2/M phase transition of the cell cycle.

    abstract::Cdc25B is a dual specificity phosphatase involved in the control of cyclin-dependent kinases and the progression of cells through the cell cycle. A series of minimal domain Cdc25B constructs maintaining catalytic activity have been expressed. The structure of a minimum domain construct binding sulfate was determined a...

    journal_title:Journal of molecular biology

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    authors: Reynolds RA,Yem AW,Wolfe CL,Deibel MR Jr,Chidester CG,Watenpaugh KD

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  • The 1.2 A structure of a novel quorum-sensing protein, Bacillus subtilis LuxS.

    abstract::In bacteria, the regulation of gene expression in response to changes in cell density is called quorum sensing. The autoinducer-2 production protein LuxS, is involved in a novel quorum-sensing system and is thought to catalyse the degradation of S-ribosylhomocysteine to homocysteine and the autoinducer molecule 4,5-di...

    journal_title:Journal of molecular biology

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    authors: Ruzheinikov SN,Das SK,Sedelnikova SE,Hartley A,Foster SJ,Horsburgh MJ,Cox AG,McCleod CW,Mekhalfia A,Blackburn GM,Rice DW,Baker PJ

    更新日期:2001-10-12 00:00:00

  • Dimerisation and an increase in active site aromatic groups as adaptations to high temperatures: X-ray solution scattering and substrate-bound crystal structures of Rhodothermus marinus endoglucanase Cel12A.

    abstract::Cellulose, a polysaccharide consisting of beta-1,4-linked glucose, is the major component of plant cell walls and consequently one of the most abundant biopolymers on earth. Carbohydrate polymers such as cellulose are molecules with vast diversity in structure and function, and a multiplicity of hydrolases operating i...

    journal_title:Journal of molecular biology

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    authors: Crennell SJ,Cook D,Minns A,Svergun D,Andersen RL,Nordberg Karlsson E

    更新日期:2006-02-10 00:00:00

  • Increased thermostability and fidelity of DNA synthesis of wild-type and mutant HIV-1 group O reverse transcriptases.

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    journal_title:Journal of molecular biology

    pub_type: 杂志文章


    authors: Alvarez M,Matamoros T,Menéndez-Arias L

    更新日期:2009-10-02 00:00:00

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    abstract::Colicin endonucleases (DNases) are bound and inactivated by immunity (Im) proteins. Im proteins are broadly cross-reactive yet specific inhibitors binding cognate and non-cognate DNases with K(d) values that vary between 10(-4) and 10(-14) M, characteristics that are explained by a 'dual-recognition' mechanism. In thi...

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    authors: Keeble AH,Joachimiak LA,Maté MJ,Meenan N,Kirkpatrick N,Baker D,Kleanthous C

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    journal_title:Journal of molecular biology

    pub_type: 杂志文章


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    journal_title:Journal of molecular biology

    pub_type: 杂志文章


    authors: Niraula TN,Haraoka K,Ando Y,Li H,Yamada H,Akasaka K

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  • The three-dimensional structure of N-succinyldiaminopimelate aminotransferase from Mycobacterium tuberculosis.

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    journal_title:Journal of molecular biology

    pub_type: 杂志文章


    authors: Weyand S,Kefala G,Weiss MS

    更新日期:2007-03-30 00:00:00

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    journal_title:Journal of molecular biology

    pub_type: 杂志文章


    authors: Cribbs DL,Leung J,Newton CH,Hayashi S,Miller RC Jr,Tener GM

    更新日期:1987-10-05 00:00:00

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    abstract::Calmodulin (CaM) is an important regulator of Kv7.x (KCNQx) voltage-gated potassium channels. Channels from this family produce neuronal M currents and cardiac and auditory I(KS) currents and harbor mutations that cause arrhythmias, epilepsy, and deafness. Despite extensive functional characterization, biochemical and...

    journal_title:Journal of molecular biology

    pub_type: 杂志文章


    authors: Xu Q,Chang A,Tolia A,Minor DL Jr

    更新日期:2013-01-23 00:00:00