The Mode of Action of Centrin

The recent finding of an interaction between calmodulin (CaM) and the tobacco mitogen-activated protein kinase phosphatase-1 (NtMKP1) establishes an important connection between Ca 2؉ signaling and the MAPK cascade, two of the most important signaling pathways in plant cells. Here we have used different biophysical techniques, including fluorescence and NMR spectroscopy as well as microcalorimetry, to characterize the binding of soybean CaM isoforms, SCaM-1 and -4, to synthetic peptides derived from the CaM binding domain of NtMKP1. We find that the actual CaM binding region is shorter than what had previously been suggested. Moreover, the peptide binds to the SCaM C-terminal domain even in the absence of free Ca 2؉ with the single Trp residue of the NtMKP1 peptides buried in a solvent-inaccessible hydrophobic region. In the presence of Ca 2؉ , the peptides bind first to the C-terminal lobe of the SCaMs with a nanomolar affinity, and at higher peptide concentrations, a second peptide binds to the N-terminal domain with lower affinity. Thermodynamic analysis demonstrates that the formation of the peptide-bound complex with the Ca 2؉ -loaded SCaMs is driven by favorable binding enthalpy due to a combination of hydrophobic and electrostatic interactions. Experiments with CaM proteolytic fragments showed that the two domains bind the peptide in an independent manner. To our knowledge, this is the first report providing direct evidence for sequential binding of two identical peptides of a target protein to CaM. Discussion of the potential biological role of this interaction motif is also provided.

Section: Ntmkp1-bsupporting

confidence: 47%

Calcium-dependent and -independent Binding of Soybean Calmodulin Isoforms to the Calmodulin Binding Domain of Tobacco MAPK Phosphatase-1

Rainaldi

Yamniuk

Murase

et al. 2007

“…Because the concentration of Ca 2ϩ is ϳ2.5 mM in human serum, these results suggest that about half of the SrtA molecules on the surface of S. aureus are Ca 2ϩ -bound during bacteremia. However, this is likely an underestimate because the binding of the substrate and ion to the protein presumably forms a closed thermodynamic cycle, which necessitates that the substrate-bound enzyme exhibit ϳ4-fold higher affinity for Ca 2ϩ as compared with the substrate-free enzyme (49). Ca 2ϩ Does Not Directly Interact with the Sorting Signal-NMR, x-ray, and targeted mutagenesis studies have localized the sorting signal binding site to a large hydrophobic surface immediately adjacent to the ion-binding pocket (18,22), raising the possibility that direct ion-substrate interactions stimulate catalysis.…”

Section: Camentioning

confidence: 99%

Staphylococcus aureus Sortase A Transpeptidase

Naik

Suree

Ilangovan

et al. 2006

Many virulence factors in GramSurface proteins on bacteria are frequently virulence factors, promoting bacterial adhesion, resistance to phagocytic killing, and host cell invasion during infection. In Gram-positive bacteria these proteins are often covalently anchored to the cell wall by sortase enzymes, a family of novel cysteine transpeptidases (1-3). The sortase A protein (SrtA) 2 from Staphylococcus aureus has been characterized extensively (4) and anchors proteins bearing a cell wall sorting signal that consists of a conserved LPXTG motif (where X is any amino acid), a hydrophobic domain, and a tail of mostly positively charged residues (4 -6). SrtA cleaves in between the threonine and glycine of the LPXTG motif (7) and catalyzes the formation of a peptide bond between the carboxylgroup of the threonine and the amine-group of the cell-wall precursor lipid II (7-9). The lipid II-linked protein is then incorporated into the peptidoglycan of the cell wall via the transglycosylation and transpeptidation reactions of bacterial cell-wall synthesis. Sortases represent an attractive target for new anti-infective agents, because they are widely distributed among a variety of bacterial pathogens (10, 11) (e.g. Bacillus anthracis, Listeria monocytogenes, Streptococcus pneumoniae, and Streptococcus pyogenes), and have been shown to be required for virulence (12-16).The catalytic domain of SrtA (SrtA ⌬N59 , residues 60 -206) adopts a conserved eight-stranded ␤-barrel fold (17, 18). The active site is organized around the catalytically essential side chain of Cys-184, whose thiolate nucleophilically attacks the threonine carbonyl carbon within the LPXTG sorting signal, forming a thioester linkage between the enzyme and substrate (19). In addition to Cys-184, the hydrophilic side chains of His-120 and Arg-197 are absolutely required for catalysis (20 -22). These residues likely participate in general acid/base catalysis, and one of them must activate the thiol for nucleophilic attack, because it is protonated at neutral pH (23). The indole ring of Trp-194 partially shields the cysteine thiol from the solvent, and its mutation to alanine reduces enzyme activity 4-fold through an unknown mechanism (20). Using NMR and crystallography, the LPXTG sorting signal binding site has recently been localized to a surface formed by strands ␤4 and ␤7, and to a proximal loop that connects strands ␤6 to ␤7(the ␤6/␤7 loop) (18,22). Substrate binding may occur through an induced-fit mechanism involving conformational changes in the ␤6/␤7 loop, because it is disordered in the absence of the sorting signal substrate (17,18). Ca 2ϩ stimulates the activity of SrtA ⌬N59 in vitro (17) and may enable S. aureus to increase the rate of surface protein anchoring as it encounters elevated concentrations of this ion at sites of infection. Because many surface proteins function as virulence factors, the stimulatory effect of Ca 2ϩ likely plays an important role in the infection process. Previously we showed that Ca 2ϩ bound to an ordered pocket positio...

“…Structural details of the C-terminal domain of Chlamydomonas reinhardtii caltractin and a 19-residue peptide from Kar1p protein, a component of spindle pole bodies required for cell integrity in yeast, have been elucidated using NMR methods (24). The formation of this complex is dependent on Ca 2ϩ binding (25). Another solution structure of a HsCen-2 fragment (26), including residues Met 84 -Trp 172 shows that a helical portion of the N-terminal domain lies within a hydrophobic binding cavity of the C-terminal domain.…”

Section: Human Centrin-2 (Hscen-2)mentioning

confidence: 99%

The Structure of the Human Centrin 2-Xeroderma Pigmentosum Group C Protein Complex

Thompson

Ryan

Salisbury

et al. 2006

117

Human centrin-2 plays a key role in centrosome function and stimulates nucleotide excision repair by binding to the xeroderma pigmentosum group C protein. To determine the structure of human centrin-2 and to develop an understanding of molecular interactions between centrin and xeroderma pigmentosum group C protein, we characterized the crystal structure of calcium-loaded full-length centrin-2 complexed with a xeroderma pigmentosum group C peptide. Our structure shows that the carboxyl-terminal domain of centrin-2 binds this peptide and two calcium atoms, whereas the amino-terminal lobe is in a closed conformation positioned distantly by an ordered ␣-helical linker. A stretch of the amino-terminal domain unique to centrins appears disordered. Two xeroderma pigmentosum group C peptides both bound to centrin-2 also interact to form an ␣-helical coiled-coil. The interface between centrin-2 and each peptide is predominantly nonpolar, and key hydrophobic residues of XPC have been identified that lead us to propose a novel binding motif for centrin.Human centrin-2 (HsCen-2) 2 is a Ca 2ϩ -binding protein of the calmodulin-parvalbumin EF-hand superfamily (2, 3). HsCen-2 and two other human centrins (4) are best known for functions outside the nucleus. Centrins have essential roles in the duplication and segregation of microtubule organizing centers (5, 6). Much research has focused on these functions, because abnormal centrosome duplication may lead to chromosomal instability and then cancer, an idea supported by discovery of supernumerary abnormal centrosomes in different human tumor cells (7-10). In addition to its role in centrosome function, HsCen-2 is found as a stabilizing component of xeroderma pigmentosum complement protein C (XPC) and HRad23B complexes (11,12). The XPCcontaining heterotrimer is involved in recognition of DNA lesions and initiation of global genome nucleotide excision repair. Global genome nucleotide excision repair is an important DNA repair pathway for damage caused by UV radiation, carcinogens, and chemotherapeutic agents, and impairment of XPC function is associated with the genetic disorder xeroderma pigmentosum. HsCen-2 appears to promote DNA binding by XPC both in vivo and in vitro and increases the specificity of the heterotrimer for damaged DNA (12, 13). The mechanism by which HsCen-2 binds to XPC is not understood.Centrins are also involved in cilia function (14). Moreover, Ca 2ϩ -triggered assembly of HsCen-1 and transducin appears to regulate transducin translocation through the connecting cilium of vertebrate photoreceptors (15)(16)(17)(18). Whereas the first centrin, also known as caltractin (Cdc31p), was found in fibers linking the flagellar apparatus to the nuclei of Tetraselmis striata green alga (19), homologs have since been identified in many organisms, including fungi, plants, and higher eukaryotes. Functional similarities between centrins from various species seem likely. For example, Ca 2ϩ -induced contractions of a fiber formed by caltractin and Sfi1p are thought to play ...