Structures of Calmodulin and a Functional Myosin Light Chain Kinase in the Activated Complex:  A Neutron Scattering Study

Krueger, Joanna K.; Olah, Glenn A.; Rokop, S. E.; Zhi, Gang; Trewhella, Jill

doi:10.1021/bi9702703

Cited by 77 publications

(73 citation statements)

References 33 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…A nominal resolution of the data is 1.2 nm, and details of the tertiary structure can obviously not be validated experimentally at this level of resolution, which is why we restricted ourselves to rigid body modeling. Solution scattering is rather sensitive to rigid body movements of structural domains and has successfully been used to model the quaternary structure of proteins (15)(16)(17). The use of rigid body modeling is justified by a high sequence homology between monomeric ␣B-crystallin and MjHSP16.5.…”

Section: Discussionmentioning

confidence: 99%

A Novel Quaternary Structure of the Dimeric α-Crystallin Domain with Chaperone-like Activity

Feil¹,

Malfois²,

Hendle³

et al. 2001

Journal of Biological Chemistry

105

View full text Add to dashboard Cite

␣B-crystallin, a member of the small heat-shock protein family and a major eye lens protein, is a high molecular mass assembly and can act as a molecular chaperone. We report a synchrotron radiation x-ray solution scattering study of a truncation mutant from the human ␣B-crystallin (␣B57-157), a dimeric protein that comprises the ␣-crystallin domain of the ␣B-crystallin and retains a significant chaperone-like activity. According to the sequence analysis (more than 23% identity), the monomeric fold of the ␣-crystallin domain should be close to that of the small heat-shock protein from Methanococcus jannaschii (MjHSP16.5). The theoretical scattering pattern computed from the crystallographic model of the dimeric MjHSP16.5 deviates significantly from the experimental scattering by the ␣-crystallin domain, pointing to different quaternary structures of the two proteins. A rigid body modeling against the solution scattering data yields a model of the ␣-crystallin domain revealing a new dimerization interface. The latter consists of a strand-turn-strand motif contributed by each of the monomers, which form a four-stranded, antiparallel, intersubunit composite ␤-sheet. This model agrees with the recent spin labeling results and suggests that the ␣B-crystallin is composed by flexible building units with an extended surface area. This flexibility may be important for biological activity and for the formation of ␣B-crystallin complexes of variable sizes and compositions.␣A-and ␣B-crystallin, which share 54% amino acid sequence identity, build the subunits of ␣-crystallin, a major eye lens protein, comprising up to 40% of the total lens proteins. The structural function of the ␣-crystallin is to assist in maintaining transparency in the lens (1). The chaperone-like function of ␣B-crystallin helps to avoid formation of large light-scattering aggregates and possibly helps to prevent cataract in the lens. Moreover, neurodegenerative diseases, ischemia, or multiple sclerosis lead to an overexpression of this protein, which makes it an object of special medical interest (2).The ␣-crystallin as well as other mammalian small heat-shock proteins (sHSPs)1 form large globular complexes with a diameter of about 10 -25 nm. Cryoelectron microscopy and image analysis revealed that ␣B-crystallin is a hollow spherical shell with variable quaternary structure (3), and a frequent exchange of subunits between the particles was observed. The chaperone activity of ␣B-crystallin is associated with partial perturbation of the substrate protein tertiary structure, leading to a multimeric molten globule-like state with increased hydrophobicity (4, 5). The exposed hydrophobic regions of ␣-crystallin interact with substrate proteins possessing an increased surface hydrophobicity but a low degree of unfolding (6).The molecular structure and subunit interactions in ␣-crystallin have long been under investigation. The stretches of residues promoting formation of lower or higher molecular weight ␣-crystallin oligomers have been identified. Upon additio...

show abstract

Section: Discussionmentioning

confidence: 99%

A Novel Quaternary Structure of the Dimeric α-Crystallin Domain with Chaperone-like Activity

Feil¹,

Malfois²,

Hendle³

et al. 2001

Journal of Biological Chemistry

105

View full text Add to dashboard Cite

show abstract

“…This can occur rapidly: Ca 2ϩ ⅐CaM activates myosin light chain kinase at about the same rate as the two proteins associate (10 7 M Ϫ1 s Ϫ1 ) (42). A recent neutron diffraction study provides evidence that CaM indeed activates myosin light chain kinase by "inducing a significant movement of the kinase's CaM binding and autoinhibitory sequences away from the surface of the catalytic core" (50).…”

Section: Discussionmentioning

confidence: 99%

“…We designed the experiments so that initially, most of the peptide was bound to vesicles, and after mixing with Ca 2ϩ ⅐CaM, most of the peptide was bound to Ca 2ϩ -calmodulin. Control experiments showed that the relaxation time for MARCKS-(151-175) transfer from the membrane to CaM ( CaM ) was independent of both lipid concentration (for example, 50 (open symbols) labeled on the first and ninth residues of the peptide, respectively. We obtain the association rate constant for a peptide binding to a vesicle by multiplying the slope by ; if ϭ 4 ϫ 10 5 , k on Ϸ 3 ϫ 10 11 M Ϫ1 s Ϫ1 for LUV 600 (Table I).…”

Section: Calmodulin-induced Dissociation Of Marcks-(151-175) Frommentioning

confidence: 99%

Kinetics of Interaction of the Myristoylated Alanine-rich C Kinase Substrate, Membranes, and Calmodulin

Arbuzova

Wang

Murray

et al. 1997

Journal of Biological Chemistry

View full text Add to dashboard Cite

Membrane binding of the myristoylated alanine-rich C kinase substrate (MARCKS) requires both its myristate chain and basic "effector" region. Previous studies with a peptide corresponding to the effector region, MARCKS-(151-175), showed that the 13 basic residues interact electrostatically with acidic lipids and that the 5 hydrophobic phenylalanine residues penetrate the polar head group region of the bilayer. Here we describe the kinetics of the membrane binding of fluorescent (acrylodan-labeled) peptides measured with a stopped-flow technique. Even though the peptide penetrates the polar head group region, the association of MARCKS-(151-175) with membranes is extremely rapid; association occurs with a diffusion-limited association rate constant. For example, k on ‫؍‬ 10 11 M ؊1 s ؊1 for the peptide binding to 100-nm diameter phospholipid vesicles. As expected theoretically, k on is independent of factors that affect the molar partition coefficient, such as the mole fraction of acidic lipid in the vesicle and the salt concentration. The dissociation rate constant (k off ) is ϳ10 s ؊1 (lifetime ‫؍‬ 0.1 s) for vesicles with 10% acidic lipid in 100 mM KCl. Ca 2؉ -calmodulin (Ca 2؉ ⅐CaM) decreases markedly the lifetime of the peptide on vesicles, e.g. from 0.1 to 0.01 s in the presence of 5 M Ca 2؉ ⅐CaM. Our results suggest that Ca 2؉ ⅐CaM collides with the membrane-bound MARCKS-(151-175) peptide and pulls the peptide off rapidly. We discuss the biological implications of this switch mechanism, speculating that an increase in the level of Ca 2؉ -calmodulin could rapidly release phosphatidylinositol 4,5-bisphosphate that previous work has suggested is sequestered in lateral domains formed by MARCKS and MARCKS-(151-175).The myristoylated alanine-rich C kinase substrate (MARCKS), 1 a major substrate of protein kinase C in many cell types, is essential for brain development (1). Although the exact function of MARCKS is not known, this peripheral protein binds to calmodulin (2) and actin (3) and may play a role in secretion, membrane trafficking, and cell motility (reviewed in Refs. 4 and 5). In the plasma membranes of macrophages, MARCKS is concentrated in lateral domains (6): it colocalizes with actin filaments and protein kinase C-␣ in nascent phagosomes (7). Binding of MARCKS to either phospholipid vesicles or biological membranes requires both the insertion of its myristate chain into the hydrocarbon interior of the membrane and interaction of its basic effector region with acidic phospholipids (Refs. 8 -13; reviewed in Refs. 14 -16). Phosphorylation by protein kinase C (reviewed in Ref. 17) introduces three negatively charged phosphates into the effector region, which weakens its electrostatic interaction with acidic lipids and produces desorption of MARCKS from membranes (9 -11, 18). Ca 2ϩ -calmodulin (Ca 2ϩ ⅐CaM) binds with high (nanomolar) affinity to the effector region, producing desorption of MARCKS from both phospholipid vesicles and plasma membranes (10, 11).Several experiments suggest that a peptide corre...

show abstract

“…Far more detailed models can be constructed when highresolution structures of individual domains or subunits composing the complex are available. Assuming that the tertiary structure of the domains is largely preserved upon the complex formation, rigid-body modelling can be used to build the macromolecular complexes (Ashton et al, 1997;Krueger et al, 1997;Svergun, Aldag et al, 1998). Thus, for an assembly of two subunits, the complex is constructed by varying six positional parameters describing the relative position and orientation of the second subunit with respect to the ®rst one.…”

Section: Introductionmentioning

confidence: 99%

MASSHA– a graphics system for rigid-body modelling of macromolecular complexes against solution scattering data

2001

View full text Add to dashboard Cite

A program, MASSHA, for three-dimensional rendering and rigid-body re®nement is presented. The program allows display and manipulation of high-resolution atomic structures and low-resolution models represented as smooth envelopes or ensembles of beads. MASSHA is coupled with a computational module to align structural models of different nature and resolution automatically. The main feature is the possibility for rigid-body re®nement of the quaternary structure of macromolecular complexes. If high-or low-resolution models of individual subunits are available, the complex can be constructed on the computer display and re®ned to ®t the experimental solution scattering data. MASSHA provides both interactive and automated re®nement modes to position subunits in a heterodimeric complex (general case) and in a homodimeric complex with a twofold symmetry axis. Examples of application of the program (running on IBM PC compatible machines under Windows 9x/NT/ 2000) are given.

show abstract

Structures of Calmodulin and a Functional Myosin Light Chain Kinase in the Activated Complex: A Neutron Scattering Study

Cited by 77 publications

References 33 publications

A Novel Quaternary Structure of the Dimeric α-Crystallin Domain with Chaperone-like Activity

A Novel Quaternary Structure of the Dimeric α-Crystallin Domain with Chaperone-like Activity

Kinetics of Interaction of the Myristoylated Alanine-rich C Kinase Substrate, Membranes, and Calmodulin

MASSHA– a graphics system for rigid-body modelling of macromolecular complexes against solution scattering data

Contact Info

Product

Resources

About