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Mark W Maciejewski

Publications and source records attributed to Mark W Maciejewski.

12 recordsLinked to original sources
PubMed2006

Mapping the phosphoinositide-binding site on chick cofilin explains how PIP2 regulates the cofilin-actin interaction.

Cofilin plays a key role in the choreography of actin dynamics via its ability to sever actin filaments and increase the rate of monomer dissociation from pointed ends. The exact manner by which phosphoinositides bind to cofilin and inhibit its interaction with actin has proven difficult to ascertain. We determined the structure of chick cofilin and used NMR chemical shift mapping and structure-directed mutagenesis to unambiguously locate its recognition site for phosphoinositides (PIs). This structurally unique recognition site requires both the acyl chain and head group of the PI for a productive interaction, and it is not inhibited by phosphorylation of cofilin. We propose that the interaction of cofilin with membrane-bound PIs abrogates its binding to both actin and actin-interacting protein 1, and facilitates spatiotemporal regulation of cofilin activity.

Actin Depolymerizing Factors↗
PubMed2006

Regulation of RhoGEF activity by intramolecular and intermolecular SH3 domain interactions.

RhoGEFs are central controllers of small G-proteins in cells and are regulated by several mechanisms. There are at least 22 human RhoGEFs that contain SH3 domains, raising the possibility that, like several other enzymes, SH3 domains control the enzymatic activity of guanine nucleotide exchange factor (GEF) domains through intra- and/or intermolecular interactions. The structure of the N-terminal SH3 domain of Kalirin was solved using NMR spectroscopy, and it folds much like other SH3 domains. However, NMR chemical shift mapping experiments showed that this Kalirin SH3 domain is unique, containing novel cooperative binding site(s) for intramolecular PXXP ligands. Intramolecular Kalirin SH3 domain/ligand interactions, as well as binding of the Kalirin SH3 domain to the adaptor protein Crk, inhibit the GEF activity of Kalirin. This study establishes a novel molecular mechanism whereby intramolecular and intermolecular Kalirin SH3 domain/ligand interactions modulate GEF activity, a regulatory mechanism that is likely used by other RhoGEF family members.

Amino Acid Motifs↗
PubMed2006

Minimotif Miner: a tool for investigating protein function.

In addition to large domains, many short motifs mediate functional post-translational modification of proteins as well as protein-protein interactions and protein trafficking functions. We have constructed a motif database comprising 312 unique motifs and a web-based tool for identifying motifs in proteins. Functional motifs predicted by MnM can be ranked by several approaches, and we validated these scores by analyzing thousands of confirmed examples and by confirming prediction of previously unidentified 14-3-3 motifs in EFF-1.

14-3-3 Proteins↗
PubMed2006

Levels of glycine betaine in growing cells and spores of Bacillus species and lack of effect of glycine betaine on dormant spore resistance.

Bacteria of various Bacillus species are able to grow in media with very high osmotic strength in part due to the accumulation of low-molecular-weight osmolytes such as glycine betaine (GB). Cells of Bacillus species grown in rich and minimal media contained low levels of GB, but GB levels were 4- to 60-fold higher in cells grown in media with high salt. GB levels in Bacillus subtilis cells grown in minimal medium were increased approximately 7-fold by GB in the medium and 60-fold by GB plus high salt. GB was present in spores of Bacillus species prepared in media with or without high salt but at lower levels than in comparable growing cells. With spores prepared in media with high salt, GB levels were highest in B. subtilis spores and > or =20-fold lower in B. cereus and B. megaterium spores. Although GB levels in B. subtilis spores were elevated 15- to 30-fold by GB plus high salt in sporulation media, GB levels did not affect spore resistance. GB levels were similar in wild-type B. subtilis spores and spores that lacked major small, acid-soluble spore proteins but were much lower in spores that lacked dipicolinic acid.

Adaptation, Physiological↗
PubMed2005

Solution structure of the Tctex1 dimer reveals a mechanism for dynein-cargo interactions.

Tctex1 is a light chain found in both cytoplasmic and flagellar dyneins and is involved in many fundamental cellular activities, including rhodopsin transport within photoreceptors, and may function in the non-Mendelian transmission of t haplotypes in mice. Here, we present the NMR solution structure for the Tctex1 dimer from Chlamydomonas axonemal inner dynein arm I1. Structural comparisons reveal a strong similarity with the LC8 dynein light chain dimer, including formation of a strand-switched beta sheet interface. Analysis of the Tctex1 structure enables the dynein intermediate chain binding site to be identified and suggests a mechanism by which cargo proteins might be attached to this microtubule motor complex. Comparison with the alternate dynein light chain rp3 reveals how the specificity of dynein-cargo interactions mediated by these dynein components is achieved. In addition, this structure provides insight into the consequences of the mutations found in the t haplotype forms of this protein.

Amino Acid Sequence↗
PubMed2004

Structure and mechanism of action of Sda, an inhibitor of the histidine kinases that regulate initiation of sporulation in Bacillus subtilis.

Histidine kinases are used extensively in prokaryotes to monitor and respond to changes in cellular and environmental conditions. In Bacillus subtilis, sporulation-specific gene expression is controlled by a histidine kinase phosphorelay that culminates in phosphorylation of the Spo0A transcription factor. Sda provides a developmental checkpoint by inhibiting this phosphorelay in response to DNA damage and replication defects. We show that Sda acts at the first step in the relay by inhibiting autophosphorylation of the histidine kinase KinA. The structure of Sda, which we determined using NMR, comprises a helical hairpin. A cluster of conserved residues on one face of the hairpin mediates an interaction between Sda and the KinA dimerization/phosphotransfer domain. This interaction stabilizes the KinA dimer, and the two proteins form a stable heterotetramer. The data indicate that Sda forms a molecular barricade that inhibits productive interaction between the catalytic and phosphotransfer domains of KinA.

Amino Acid Sequence↗
PubMed2004

Modulation of agrin function by alternative splicing and Ca2+ binding.

The aggregation of acetylcholine receptors on postsynaptic membranes is a key step in neuromuscular junction development. This process depends on alternatively spliced forms of the proteoglycan agrin with "B-inserts" of 8, 11, or 19 residues in the protein's globular C-terminal domain, G3. Structures of the neural B8 and B11 forms of agrin-G3 were determined by X-ray crystallography. The structure of G3-B0, which lacks inserts, was determined by NMR. The agrin-G3 domain adopts a beta jellyroll fold. The B insert site is flanked by four loops on one edge of the beta sandwich. The loops form a surface that corresponds to a versatile interaction interface in the family of structurally related LNS proteins. NMR and X-ray data indicate that this interaction interface is flexible in agrin-G3 and that flexibility is reduced by Ca(2+) binding. The plasticity of the interaction interface could enable different splice forms of agrin to select between multiple binding partners.

Agrin↗
PubMed2003

Relaxation-based structure refinement and backbone molecular dynamics of the dynein motor domain-associated light chain.

The light chain 1 (LC1) polypeptide is a member of the leucine-rich repeat protein family and binds at or near the ATP hydrolytic site within the motor domain of the gamma heavy chain from Chlamydomonas outer arm dynein. It consists of an N-terminal helix, a central barrel formed from six leucine-rich repeats that fold as beta beta alpha units, and a C-terminal helical domain that protrudes from the main axis defined by the leucine-rich repeats. Interaction with the gamma heavy chain is likely mediated through a hydrophobic patch on the larger beta sheet face, and the C-terminal region is predicted to insert into the dynein ATP hydrolytic site. Here we have used 1H-15N heteronuclear relaxation measurements obtained at 500 and 600 MHz to refine and validate the LC1 solution structure. In this refined structure, the C-terminal helix is significantly reoriented by more than 20 degrees as compared to the control and provides a more precise understanding of the potential regulatory role of this domain. We also employed the refined structure to perform a dynamic analysis of LC1 using the 600 MHz data set. These results, which were cross validated using the 500 MHz data set, strongly support identification of the predicted LC1 binding surfaces and provide additional insight into the interaction mechanisms of leucine-rich repeat proteins.

Animals↗
PubMed2002

Mapping of the interaction interface of DNA polymerase beta with XRCC1.

Residues of DNA polymerase beta (beta-Pol) that interact with the DNA repair protein XRCC1 have been determined by NMR chemical shift mapping (CSM) and mutagenesis. 15N/(13)C/(2)H/(1)H,(13)C-methyl(Leu,Ile,Val)-labeled beta-Pol palm-thumb domain was used for assignments of the 1H, 15N, and 13C resonances used for CSM of the palm-thumb on forming the 40 kDa complex with the XRCC1 N-terminal domain (NTD). Large chemical shift changes were observed in the thumb on complexation. 15N relaxation data indicate reduction in high-frequency motion for a thumb loop and three palm turn/loops, which showed concomitant chemical shift changes on complexation. A deltaV303-V306 deletion and an L301R/V303R/V306R triple mutation abolished complex formation due to loss in hydrophobicity. In an updated model, the thumb-loop of beta-Pol contacts an edge/face region of the beta sheet of the XRCC1 NTD, while the beta-Pol palm weakly contacts the alpha2 helix.

Amino Acid Sequence↗