Mapping the Binding Site for Matrix Metalloproteinase on the N-Terminal Domain of the Tissue Inhibitor of Metalloproteinases-2 by NMR Chemical Shift Perturbation

Williamson, Richard A.; Carr, Mark D.; Frenkiel, Thomas A.; Feeney, J.; Freedman, Robert B.

doi:10.1021/bi9712091

Cited by 121 publications

(123 citation statements)

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“…S3B). The residues of FusB involved in the interaction between FusB and EF-G were identified by chemical shift mapping of minimal chemical shift changes (17,18), and were found to reside almost exclusively in the C-terminal portion of the protein (Fig. 2 A and C).…”

Section: Resultsmentioning

confidence: 99%

“…Chemical shift indexing (CSI) (16) was used to determine the secondary structure of FusB from the shifts of 1 H and 13 C nuclei. Conservative chemical shift differences between the 1 H -15 N spectra for FusB and FusB·EF-G C3 were calculated by finding the closest peak in the FusB·EF-G C3 spectrum to the assigned peaks in the FusB spectrum (18). Shift differences for which Δ >0.6 ppm were considered significant and indicated residues involved in forming the FusB·EF-G C3 interface.…”

Section: Methodsmentioning

confidence: 99%

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Ribosome clearance by FusB-type proteins mediates resistance to the antibiotic fusidic acid

Cox

Thompson

Jenkins

et al. 2012

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

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Resistance to the antibiotic fusidic acid (FA) in the human pathogen Staphylococcus aureus usually results from expression of FusBtype proteins (FusB or FusC). These proteins bind to elongation factor G (EF-G), the target of FA, and rescue translation from FAmediated inhibition by an unknown mechanism. Here we show that the FusB family are two-domain metalloproteins, the C-terminal domain of which contains a four-cysteine zinc finger with a unique structural fold. This domain mediates a high-affinity interaction with the C-terminal domains of EF-G. By binding to EF-G on the ribosome, FusB-type proteins promote the dissociation of stalled ribosome·EF-G·GDP complexes that form in the presence of FA, thereby allowing the ribosomes to resume translation. Ribosome clearance by these proteins represents a highly unusual antibiotic resistance mechanism, which appears to be fine-tuned by the relative abundance of FusB-type protein, ribosomes, and EF-G.antibacterial | protein synthesis | translocation | structure

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Ribosome clearance by FusB-type proteins mediates resistance to the antibiotic fusidic acid

Cox

Thompson

Jenkins

et al. 2012

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

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“…In TIMP-2, this region is extended by 7 residues compared with TIMP-1 and is therefore capable of making more extensive interactions with the proteinase. The involvement of the extended AB ␤-hairpin in TIMP-2/MMP interactions was first proposed on the basis of chemical shift changes observed on binding of N-TIMP-2 to N-MMP-3 (16), and further confirmed in the crystal structure for the TIMP-2⅐MT1-MMP complex (15).…”

mentioning

confidence: 90%

“…Crystal structures have also been published for full-length TIMP-1 and TIMP-2 in complexes with the catalytic domains of MMP-3 (N-MMP-3) and MT1-MMP, respectively (14,15). The structures of the TIMP⅐MMP complexes, together with NMR data on chemical shift perturbation seen for N-TIMP-2 on complex formation with N-MMP-3 (16), have identified the key features of the TIMP inhibitory binding site. It is now clear that the N terminus of TIMP (residues 1-5), together with the two loops with which it is disulfide-bonded, form a "wedge"-like structure that interacts with the active site cleft of the proteinase.…”

mentioning

confidence: 99%

“…These studies provided some insights into the dynamics of N-TIMP-2, particularly for the AB ␤-hairpin, which we proposed to be a highly flexible structure due to the relatively narrow line widths seen for signals in this region and the lack of long range NOEs. Furthermore, we suggested that another region of the N-TIMP-2 binding site (Ser 68 -Cys 72 ) was in relatively slow exchange between multiple conformations interconverting on a millisecond time scale, as the backbone amide resonances for these residues were missing from NMR spectra (16).…”

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confidence: 99%

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The Effect of Matrix Metalloproteinase Complex Formation on the Conformational Mobility of Tissue Inhibitor of Metalloproteinases-2 (TIMP-2)

Williamson

Muskett

Howard

et al. 1999

Journal of Biological Chemistry

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The backbone mobility of the N-terminal domain of tissue inhibitor of metalloproteinases-2 (N-TIMP-2) was determined both for the free protein and when bound to the catalytic domain of matrix metalloproteinase-3 (N-MMP-3). Regions of the protein with internal motion were identified by comparison of the T 1 and T 2 relaxation times and 1 H-15 N nuclear Overhauser effect values for the backbone amide 15 N signals for each residue in the sequence. This analysis revealed rapid internal motion on the picosecond to nanosecond time scale for several regions of free N-TIMP-2, including the extended ␤-hairpin between ␤-strands A and B, which forms part of the MMP binding site. Evidence of relatively slow motion indicative of exchange between two or more local conformations on a microsecond to millisecond time scale was also found in the free protein, including two other regions of the MMP binding site (the CD and EF loops). On formation of a tight N-TIMP-2⅐N-MMP-3 complex, the rapid internal motion of the AB ␤-hairpin was largely abolished, a change consistent with tight binding of this region to the MMP-3 catalytic domain. The extended AB ␤-hairpin is not a feature of all members of the TIMP family; therefore, the binding of this highly mobile region to a site distant from the catalytic cleft of the MMPs suggests a key role in TIMP-2 binding specificity.Breakdown of the extracellular matrix is an important event in many normal and pathological processes, such as growth, wound repair, tumor metastasis, and arthritis (1-3). A large family of zinc-dependent proteinases, the matrix metalloproteinases (MMPs), 1 are thought to be primarily responsible for this matrix catabolism. The activities of the MMP family in the extracellular matrix are highly regulated by transcriptional control, zymogen activation, and inhibition by a family of specific protein inhibitors, the tissue inhibitors of metalloproteinases or TIMPs (4). The TIMPs bind tightly to the active proteinases to form an inactive TIMP⅐MMP complex (5). Four mammalian TIMP proteins have now been identified (TIMP-1 to -4) (6 -9), and their high degree of sequence similarity and conservation of 12 Cys residues suggests that each consists of the same basic fold but with some variations in loop structures and glycosylation. The location of the MMP inhibitory site on the TIMP molecule has been shown to reside predominantly in the N-terminal two-thirds of the protein, defined by the first three disulfide bonds. This domain (N-TIMP) can be expressed independently to generate a fully folded, stable, and active inhibitor (10, 11).High resolution three-dimensional structures are now available for both the active N-terminal domain of TIMP-2 (12) and for the full-length inhibitor (13). Crystal structures have also been published for full-length TIMP-1 and TIMP-2 in complexes with the catalytic domains of MMP-3 (N-MMP-3) and MT1-MMP, respectively (14, 15). The structures of the TIMP⅐MMP complexes, together with NMR data on chemical shift perturbation seen for N-TIMP-2 on complex fo...

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Structural and Dynamic Information on Ligand Binding

Roberts¹

2011

Protein NMR Spectroscopy: Practical Techniques and Applications

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The function of the vast majority of proteins in the cell depends upon their noncovalent interactions with other molecules, ranging from enzyme-substrate interactions to the array of protein-protein and protein-nucleic acid interactions involved in, for example, the regulation of transcription. NMR has proved very valuable in the study of the interaction of proteins with both small and large molecules. In this chapter the focus will be on the basic principles of the study of ligand binding by NMR and on applications to small moleculeprotein interactions. Studies of macromolecular complexes are covered in Chapter 8. The use of NMR to study protein-ligand interactions is a vast area and, for reasons of space, many of the references herein are to reviews rather than to original papers; my apologies to those whose important contributions have not been cited directly.NMR can of course provide information on the structure of protein-ligand complexes, just as it can on the structure of the protein itself. However, the NMR spectrum is sensitive to both the equilibrium and the kinetics of the binding process. These effects can provide valuable information in themselves but, most importantly, they must be understood in order to interpret the spectrum correctly.

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Mapping the Binding Site for Matrix Metalloproteinase on the N-Terminal Domain of the Tissue Inhibitor of Metalloproteinases-2 by NMR Chemical Shift Perturbation

Cited by 121 publications

References 46 publications

Ribosome clearance by FusB-type proteins mediates resistance to the antibiotic fusidic acid

Ribosome clearance by FusB-type proteins mediates resistance to the antibiotic fusidic acid

The Effect of Matrix Metalloproteinase Complex Formation on the Conformational Mobility of Tissue Inhibitor of Metalloproteinases-2 (TIMP-2)

Structural and Dynamic Information on Ligand Binding

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