The solution structure of serine protease PB92 from Bacillus alcalophilus presents a rigid fold with a flexible substrate-binding site

Martin, John R.; Mulder, Frans A. A.; Karimi-Nejad, Yasmin; Zwan, Johan van der; Mariani, Matteo; Schipper, Dick; Boelens, Rolf

doi:10.1016/s0969-2126(97)00208-6

Cited by 51 publications

(36 citation statements)

References 74 publications

(138 reference statements)

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“…In addition, inhibitorfree subtilisin displays a relatively high structural flexibility at the S1 and S4 pockets (27), indicating plasticity and hence facilitating the binding of different sequence motives. In furin, the substrate-binding area is characterized by low B-factors, indicating a rigid framework in both the unliganded and the ligand-bound forms.…”

Section: Discussionmentioning

confidence: 99%

Structure of the unliganded form of the proprotein convertase furin suggests activation by a substrate-induced mechanism

Dahms

Arciniega

Steinmetzer

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

200

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Proprotein convertases (PCs) are highly specific proteases required for the proteolytic modification of many secreted proteins. An unbalanced activity of these enzymes is connected to pathologies like cancer, atherosclerosis, hypercholesterolaemia, and infectious diseases. Novel protein crystallographic structures of the prototypical PC family member furin in different functional states were determined to 1.8-2.0 Å. These, together with biochemical data and modeling by molecular dynamics calculations, suggest essential elements underlying its unusually high substrate specificity. Furin shows a complex activation mechanism and exists in at least four defined states: (i) the "off state," incompatible with substrate binding as seen in the unliganded enzyme; (ii) the active "on state" seen in inhibitor-bound furin; and the respective (iii) calcium-free and (iv) calcium-bound forms. The transition from the off to the on state is triggered by ligand binding at subsites S1 to S4 and appears to underlie the preferential recognition of the four-residue sequence motif of furin. The molecular dynamics simulations of the four structural states reflect the experimental observations in general and provide approximations of the respective stabilities. Ligation by calcium at the PC-specific binding site II influences the active-site geometry and determines the rotamer state of the oxyanion holeforming Asn295, and thus adds a second level of the activity modulation of furin. The described crystal forms and the observations of different defined functional states may foster the development of new tools and strategies for pharmacological intervention targeting furin.serine-protease | activation | specificity | conformational transition

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Section: Discussionmentioning

confidence: 99%

Structure of the unliganded form of the proprotein convertase furin suggests activation by a substrate-induced mechanism

Dahms

Arciniega

Steinmetzer

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

200

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“…In the case of the second substitution; Gly102 with Ser, the side chain of serine protrude inside the S4 pocket which seems to decrease the size of S4a site and this change also contribute to a slight change in the hydrophobicity of the S4 pocket. Even though the size of the S4 pocket in EAP is reduced by these two substitutions, it can still accommodate bulky P4 residues like PRK1, since these two substitutions are located in the highly flexible, surface exposed loops 100-104 and 132-136, respectively (Martin et al 1997). Especially, the segment 100-104 shows larger conformational freedom which confers on S4 pocket the capacity to accommodate a large P4 residue (Wolf et al 1991).…”

Section: Discussionmentioning

confidence: 99%

Molecular cloning and homology modelling of a subtilisin-like serine protease from the marine fungus, Engyodontium album BTMFS10

Corso

Chellappan

Sukumaran

et al. 2010

World J Microbiol Biotechnol

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An alkaline protease gene (Eap) was isolated for the first time from a marine fungus, Engyodontium album. Eap consists of an open reading frame of 1,161 bp encoding a prepropeptide consisting of 387 amino acids with a calculated molecular mass of 40.923 kDa. Homology comparison of the deduced amino acid sequence of Eap with other known proteins indicated that Eap encode an extracellular protease that belongs to the subtilase family of serine protease (Family S8). A comparative homology model of the Engyodontium album protease (EAP) was developed using the crystal structure of proteinase K. The model revealed that EAP has broad substrate specificity similar to Proteinase K with preference for bulky hydrophobic residues at P1 and P4. Also, EAP is suggested to have two disulfide bonds and more than two Ca 2? binding sites in its 3D structure; both of which are assumed to contribute to the thermostable nature of the protein.Keywords Engyodontium album Á Alkaline serine protease Á Subtilases Á Homology modelling Abbreviations E. album Engyodontium album EAPEngyodontium album protease (deduced protein) Eap Engyodontium album protease (gene)

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“…Thus, it is now potentially feasible to determine the structures of proteins in the 15-to 35-kDa range at a resolution comparable to Ϸ2.5-Å resolution crystal structures (7). The upper limit of applicability is probably 60-70 kDa, and the largest single-chain proteins solved to date are Ϸ30 kDa, comprising Ϸ260 residues (8,9). In this paper, we discuss a number of new refinement strategies aimed at both facilitating NMR structure determination and increasing the accuracy of the resulting structures.…”

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confidence: 99%

New methods of structure refinement for macromolecular structure determination by NMR

Clore

Gronenborn²

1998

Proc. Natl. Acad. Sci. U.S.A.

231

177

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Recent advances in multidimensional NMR methodology have permitted solution structures of proteins in excess of 250 residues to be solved. In this paper, we discuss several methods of structure refinement that promise to increase the accuracy of macromolecular structures determined by NMR. These methods include the use of a conformational database potential and direct refinement against three-bond coupling constants, secondary 13 C shifts, 1 H shifts, T 1 ͞T 2 ratios, and residual dipolar couplings. The latter two measurements provide long range restraints that are not accessible by other solution NMR parameters.The two major techniques for determining the threedimensional structures of macromolecules at atomic resolution are x-ray crystallography in the solid state (single crystals) and NMR spectroscopy in solution. Unlike crystallography, NMR measurements are not hampered by the ability or inability of a protein to crystallize. The size of macromolecular structures that can be solved by NMR has been increased dramatically over the last few years (1). The development of a wide range of two-dimensional (2D) NMR experiments in the early 1980s culminated in the determination of the structures of a number of small proteins (2, 3). Under exceptional circumstances, 2D NMR techniques can be applied successfully to determine structures of proteins up to Ϸ100 residues (4, 5). Beyond Ϸ100 residues, however, 2D NMR methods fail, principally because of spectral complexity that cannot be resolved in two dimensions. In the late 1980s and early 1990s, a series of major advances took place in which the spectral resolution was increased by extending the dimensionality to three and four dimensions (1). In addition, by combining such multidimensional experiments with heteronuclear NMR, problems associated with large linewidths can be circumvented by making use of heteronuclear couplings that are large relative to the linewidths. The first successful application of these methods to a protein greater than Ϸ12 kDa was achieved in 1991 with the determination of the solution structure of interleukin 1␤, a protein of 18 kDa and 153 residues (6). Concomitant with spectroscopic advances, significant improvements have taken place in the accuracy with which macromolecular structures can be determined. Thus, it is now potentially feasible to determine the structures of proteins in the 15-to 35-kDa range at a resolution comparable to Ϸ2.5-Å resolution crystal structures (7). The upper limit of applicability is probably 60-70 kDa, and the largest single-chain proteins solved to date are Ϸ30 kDa, comprising Ϸ260 residues (8, 9). In this paper, we discuss a number of new refinement strategies aimed at both facilitating NMR structure determination and increasing the accuracy of the resulting structures. These include direct refinement against three-bond coupling constants (10) and 13 C and 1 H shifts (11-13), as well as the use of conformational database potentials (14, 15). More recently, methods have been developed to obtain structural...

show abstract

The solution structure of serine protease PB92 from Bacillus alcalophilus presents a rigid fold with a flexible substrate-binding site

Cited by 51 publications

References 74 publications

Structure of the unliganded form of the proprotein convertase furin suggests activation by a substrate-induced mechanism

Structure of the unliganded form of the proprotein convertase furin suggests activation by a substrate-induced mechanism

Molecular cloning and homology modelling of a subtilisin-like serine protease from the marine fungus, Engyodontium album BTMFS10

New methods of structure refinement for macromolecular structure determination by NMR

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