Electrospray mass spectrometric investigation of the chaperone SecB

Smith, Virginia F.; Schwartz, Brenda L.; Randall, Linda L.; Smith, Richard

doi:10.1002/pro.5560050310

Cited by 69 publications

(54 citation statements)

References 28 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…proteins from E. coli (30,31). The difference between the experimental and theoretical mass of the protein is 0.55 Da, which is an acceptable error of the instrument.…”

Section: Downloaded Frommentioning

confidence: 82%

The Matrix Metalloproteinase 9 (MMP-9) Hemopexin Domain Is a Novel Gelatin Binding Domain and Acts as an Antagonist

Roeb¹,

Schleinkofer²,

Kernebeck³

et al. 2002

Journal of Biological Chemistry

View full text Add to dashboard Cite

Matrix metalloproteinases (MMPs) are involved in the remodeling processes of the extracellular matrix and the basement membrane. Most MMPs are composed of a regulatory, a catalytic, and a hemopexin subunit. In many tumors the expression of MMP-9 correlates with local tumor growth, invasion, and metastasis. To analyze the role of the hemopexin domain in these processes, the MMP-9 hemopexin domain (MMP-9-PEX) was expressed as a glutathione S-transferase fusion protein in Escherichia coli. After proteolytic cleavage, the isolated PEX domain was purified by size exclusion chromatography. In a zymography assay, MMP-9-PEX was able to inhibit MMP-9 activity. The association and dissociation rates for the interaction of MMP-9-PEX with gelatin were determined by plasmon resonance. From the measured rate constants, the dissociation constant was calculated to be K d ‫؍‬ 2,4 ؋ 10 ؊8 M, demonstrating a high affinity between MMP-9-PEX and gelatin. In Boyden chamber experiments the recombinant MMP-9-PEX was able to inhibit the invasion of melanoma cells secreting high amounts of MMP-9 in a dose-dependent manner. These data demonstrate for the first time that the hemopexin domain of MMP-9 has a high affinity binding site for gelatin, and the particular recombinant domain is able to block MMP-9 activity and tumor cell invasion. Because MMP-9 plays an important role in metastasis, this antagonistic effect may be utilized to design MMP inhibition-based cancer therapy.Matrix metalloproteinases (MMPs) 1 are a family of zinc metallo-endopeptidases secreted by cells. They are responsible for most of the turnover of matrix components. The MMPs are produced as zymogens with a signal sequence and propeptide segment that has to be removed during activation. The propeptide domain contains a conserved cysteine that chelates the zinc in the active site. The gelatinases MMP-2 and MMP-9 contain fibronectin type II domains that are inserted in the middle of the catalytic domain, presumably to enhance substrate binding (1). MMP-9 also has a collagen type V-like domain located between the catalytic and the C-terminal hemopexin domain (Fig. 1). All but two MMPs (MMP-7 and MMP-26) contain a regulatory subunit, the hemopexin domain, separated from the catalytic domain by a variable hinge region (2). This domain is thought to confer much of the substrate specificity to the MMPs (3). It is involved in activation as well as inhibition of MMPs (3,4) and may enhance substrate binding and specificity (5). The hinge region also confers specificity to the MMPs either by direct binding of the substrate or by setting the orientation of the hemopexin domain and the catalytic domain (6). The hemopexin domain of MMP-2 is known to bind heparin (7). Heparin has been shown to potentiate the activities of some MMPs, and MMPs are often found associated with heparin sulfate glycosaminoglycans on the cell surface (8). The overall three-dimensional structure of the hemopexin domain is a four-bladed propeller with a calcium binding site nestled in the folds (3). A fragme...

show abstract

“…proteins from E. coli (30,31). The difference between the experimental and theoretical mass of the protein is 0.55 Da, which is an acceptable error of the instrument.…”

Section: Downloaded Frommentioning

confidence: 82%

The Matrix Metalloproteinase 9 (MMP-9) Hemopexin Domain Is a Novel Gelatin Binding Domain and Acts as an Antagonist

Roeb¹,

Schleinkofer²,

Kernebeck³

et al. 2002

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…One of the first applications of structural mass spectrometry involved defining the stoichiometry of protein assemblies [28,29]. When the analyzed sample is a homo-oligomer, stoichiometry determination is quite straightforward, as the obtained mass is simply divided by the mass of the monomeric subunit to obtain the subunit copy number.…”

Section: Protein Complex Composition and Stoichiometrymentioning

confidence: 99%

How far can we go with structural mass spectrometry of protein complexes?

Sharon

2010

J. Am. Soc. Mass Spectrom.

100

View full text Add to dashboard Cite

Physical interactions between proteins and the formation of stable complexes form the basis of most biological functions. Therefore, a critical step toward understanding the integrated workings of the cell is to determine the structure of protein complexes, and reveal how their structural organization dictates function. Studying the three-dimensional organization of protein assemblies, however, represents a major challenge for structural biologists, due to the large size of the complexes, their heterogeneous composition, their flexibility, and their asymmetric structure. In the last decade, mass spectrometry has proven to be a valuable tool for analyzing such noncovalent complexes. Here, I illustrate the breadth of structural information that can be obtained from this approach, and the steps taken to elucidate the stoichiometry, topology, packing, dynamics, and shape of protein complexes. In addition, I illustrate the challenges that lie ahead, and the future directions toward which the field might be heading. (J Am Soc Mass Spectrom 2010, 21, 487-500)

show abstract

“…3, q). A truncated form of SecB (SecB141) that has lost 14 aminoacyl residues from the C terminus as a result of proteinase K cleavage at Leu-141 (29) was somewhat less active in blocking folding under these conditions (Fig. 3, OE and ).…”

Section: Potentiation Of Secb To Block Folding Of Maltose-bindingmentioning

confidence: 99%

Kinetic Partitioning

Diamond

Randall

1997

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Chaperones are a class of proteins that possess the remarkable ability to selectively bind polypeptides that are in a nonnative state. The selectivity of SecB, a molecular chaperone in Escherichia coli, for its ligands can be explained in part by a kinetic partitioning between folding of the polypeptide and association with SecB. It has clearly been established that kinetic partitioning can be poised to favor association with SecB by changing the rate constant for folding of the ligand. We now demonstrate that binding to SecB can be given a kinetic advantage over the pathway for folding by modulating the properties of the chaperone. By poising SecB to expose a hydrophobic patch, we were able to detect a complex between SecB and maltose-binding protein under conditions in which rapid folding of the polypeptide otherwise precludes formation of a kinetically stable complex. The data presented here are interpreted within the framework of a kinetic partitioning between binding to SecB and folding of the polypeptide. We propose that exposure of a hydrophobic patch on SecB increases the surface area for binding and thereby increases the rate constant for association. In this way association of SecB with the polypeptide ligand has a kinetic advantage over the pathway for folding.

show abstract

Electrospray mass spectrometric investigation of the chaperone SecB

Cited by 69 publications

References 28 publications

The Matrix Metalloproteinase 9 (MMP-9) Hemopexin Domain Is a Novel Gelatin Binding Domain and Acts as an Antagonist

The Matrix Metalloproteinase 9 (MMP-9) Hemopexin Domain Is a Novel Gelatin Binding Domain and Acts as an Antagonist

How far can we go with structural mass spectrometry of protein complexes?

Kinetic Partitioning

Contact Info

Product

Resources

About