An Epitope Proximal to the Carboxyl Terminus of the α-Subunit is Located near the Lobe Tips of the Phosphorylase Kinase Hexadecamer

Wilkinson, Deborah A.; Marion, Tony N.; Tillman, David M.; Norcum, Mona Trempe; Hainfeld, James F.; Seyer, Jerome M.; Carlson, Gerald M.

doi:10.1006/jmbi.1994.1051

Cited by 47 publications

(62 citation statements)

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“…Autophosphorylated PhK was prepared as described previously (33). The mAbs against the ␣, ␤, and ␥ subunits of PhK were previously described (15,16), and the anti-calmodulin mAb was from Zymed Laboratories Inc.. All other secondary conjugates were from Southern Biotechnology. Truncation mutants of the PhK ␤ subunit were constructed as described previously (7).…”

Section: Methodsmentioning

confidence: 99%

“…1), which has previously been shown to produce intermolecular and intramolecular cross-linked forms of the ␤ subunit with this form of the enzyme (29). Four major high molecular weight ␤-containing conjugates were identified by their apparent mass and crossreactivity against subunit-specific mAbs (15,16). With the exception of an ␣␤ dimer (mass Exp ϭ 263.6 kDa, 4.0% error), all the remaining conjugates cross-reacted only with an anti-␤ subunit-specific mAb and were consistent by mass with a ␤ 4 tetramer (mass Exp ϭ 500.8 kDa, 4.1% error) and two ␤ 2 dimers (mass Theo ϭ 250.2 kDa, 5.0% error (␤␤ x1 ); 23.3% error (␤␤ x2 )).…”

Section: Determination Of 2°structure Of the ␤ Subunit Within The Phkmentioning

confidence: 99%

“…3 kDa; 1.3% difference), with water from the solvation sphere accounting primarily for the small mass addition observed for the tetramer (67). Given the known D 2 symmetrical head-to-head packing of ␣␤␥␦ protomers in each of the two octameric lobes of PhK (15), the only possible arrangement for the ␤ subunits in the complex that can account for these results and our previous findings is a centrally located core, with the four ␤ subunits occupying a region comprising the bridges and internal lobe-faces of PhK.…”

Section: Determination Of 2°structure Of the ␤ Subunit Within The Phkmentioning

confidence: 99%

“…Reconstructions of images of PhK from EM reveal it to be a D 2 symmetrical structure of two lobes (10 -12), each composed of two ␣␤␥␦ protomers packed head-to-head with the lobes associated through interconnecting bridges (13)(14)(15)(16). The number of bridges reported for PhK from EM fields of the negatively stained complex on solid grids varies from a minimum of two to a maximum of four (13,17), with reconstructions of the stained kinase revealing only two large bridges between the lobes (10,12,18).…”

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confidence: 99%

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Structure and Location of the Regulatory β Subunits in the (αβγδ)4 Phosphorylase Kinase Complex

Nadeau

Lane

et al. 2012

Journal of Biological Chemistry

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Background: Structural information concerning the phosphorylatable regulatory ␤ subunit of phosphorylase kinase was lacking. Results: Chemical, biochemical, biophysical, and computational approaches revealed secondary, tertiary, and quaternary structures for this subunit. Conclusion: The ␤ subunit is helical and forms the ␤ 4 -bridged core in the (␣␤␥␦) 4 kinase complex. Significance: These findings reveal the architecture of the complex, which provides an explanation for the conformational changes in its bridged core associated with activating ␤-phosphorylation.

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Section: Methodsmentioning

confidence: 99%

Section: Determination Of 2°structure Of the ␤ Subunit Within The Phkmentioning

confidence: 99%

Section: Determination Of 2°structure Of the ␤ Subunit Within The Phkmentioning

confidence: 99%

mentioning

confidence: 99%

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Structure and Location of the Regulatory β Subunits in the (αβγδ)4 Phosphorylase Kinase Complex

Nadeau

Lane

et al. 2012

Journal of Biological Chemistry

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“…Its concentration was determined by methods described previously (34). The mAbs against the ␣, ␤, and ␥ subunits of PhK were described previously (35,36), and the anti-calmodulin mAb was from Zymed Laboratories Inc. All other secondary conjugates were from Southern Biotechnology.…”

Section: Methodsmentioning

confidence: 99%

CrossSearch, a User-friendly Search Engine for Detecting Chemically Cross-linked Peptides in Conjugated Proteins

Nadeau

Wyckoff

Paschall

et al. 2008

Molecular & Cellular Proteomics

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Chemical cross-linking and high resolution MS have been integrated successfully to capture protein interactions and provide low resolution structural data for proteins that are refractive to analyses by NMR or crystallography. Despite the versatility of these combined techniques, the array of products that is generated from the cross-linking and proteolytic digestion of proteins is immense and generally requires the use of labeling strategies and/or data base search algorithms to distinguish actual cross-linked peptides from the many side products of cross-linking. Most strategies reported to date have focused on the analysis of small cross-linked protein complexes (<60 kDa) because the number of potential forms of covalently modified peptides increases dramatically with the number of peptides generated from the digestion of such complexes. We report herein the development of a userfriendly search engine, CrossSearch, that provides the foundation for an overarching strategy to detect crosslinked peptides from the digests of large (>170-kDa) cross-linked proteins, i.e. conjugates. Our strategy combines the use of a low excess of cross-linker, data base searching, and Fourier transform ion cyclotron resonance MS to experimentally minimize and theoretically cull the side products of cross-linking. Using this strategy, the (␣␤␥␦) 4 phosphorylase kinase model complex was cross-linked to form with high specificity a 170-kDa ␤␥ conjugate in which we identified residues involved in the intramolecular cross-linking of the 125-kDa ␤ subunit between its regulatory N terminus and its C terminus. This finding provides an explanation for previously published homodimeric two-hybrid interactions of the ␤ subunit and suggests a dynamic structural role for the regulatory N terminus of that subunit. The results offer proof of concept for the CrossSearch strategy for analyzing conjugates and are the first to reveal a tertiary structural element of either homologous ␣ or ␤ regulatory subunit of phosphorylase kinase. Molecular & Cellular Proteomics 7:739 -749, 2008.Chemical cross-linking of proteins is a versatile technique with uses ranging from screening to obtaining relative or absolute structural information for interacting proteins (1). For large proteins that are refractive to techniques such as crystallography or NMR, cross-linking provides a means of gaining moderate to low resolution structural data by generating distance measurements between specific regions of interacting proteins (intermolecular cross-linking) or individual proteins (intramolecular cross-linking). Advances in modern MS methods have promoted a resurgence in the use of crosslinking by eliminating the need for large quantities of conjugates (term used herein to denote either proteins or peptides that are covalently cross-linked) to isolate and identify distinct regions of inter-or intramolecular cross-linked peptides (2, 3). Nanomolar amounts of conjugates are now routinely digested in gel to provide sufficient quantities of peptides for detection by MS meth...

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A model for activation of the hexadecameric phosphorylase kinase complex deduced from zero‐length oxidative crosslinking

2015

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Phosphorylase kinase (PhK) is a hexadecameric (abcd) 4 enzyme complex that upon activation by phosphorylation stimulates glycogenolysis. Due to its large size (1.3 MDa), elucidating the structural changes associated with the activation of PhK has been challenging, although phosphoactivation has been linked with an increased tendency of the enzyme's regulatory b-subunits to self-associate. Here we report the effect of a peptide mimetic of the phosphoryltable N-termini of b on the selective, zero-length, oxidative crosslinking of these regulatory subunits to form b-b dimers in the nonactivated PhK complex. This peptide stimulated b-b dimer formation when not phosphorylated, but was considerably less effective in its phosphorylated form. Because this peptide mimetic of b competes with its counterpart region in the nonactivated enzyme complex in binding to the catalytic c-subunit, we were able to formulate a structural model for the phosphoactivation of PhK. In this model, the nonactivated state of PhK is maintained by the interaction between the nonphosphorylated N-termini of b and the regulatory C-terminal domains of the c-subunits; phosphorylation of b weakens this interaction, leading to activation of the c-subunits.

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An Epitope Proximal to the Carboxyl Terminus of the α-Subunit is Located near the Lobe Tips of the Phosphorylase Kinase Hexadecamer

Cited by 47 publications

References 0 publications

Structure and Location of the Regulatory β Subunits in the (αβγδ)4 Phosphorylase Kinase Complex

Structure and Location of the Regulatory β Subunits in the (αβγδ)4 Phosphorylase Kinase Complex

CrossSearch, a User-friendly Search Engine for Detecting Chemically Cross-linked Peptides in Conjugated Proteins

A model for activation of the hexadecameric phosphorylase kinase complex deduced from zero‐length oxidative crosslinking

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