Consequences of Domain Insertion on the Stability and Folding Mechanism of a Protein

SlyD, the sensitive-to-lysis protein from Escherichia coli, consists of two domains. They are not arranged successively along the protein chain, but one domain, the “insert-in-flap” (IF) domain, is inserted internally as a guest into a surface loop of the host domain, which is a prolyl isomerase of the FK506 binding protein (FKBP) type. We used SlyD as a model to elucidate how such a domain insertion affects the stability and folding mechanism of the host and the guest domain. For these studies, the two-domain protein was compared with a single-domain variant SlyDΔIF, SlyD* without the chaperone domain (residues 1-69 and 130-165) in which the IF domain was removed and replaced by a short loop, as present in human FKBP12. Equilibrium unfolding and folding kinetics followed an apparent two-state mechanism in the absence and in the presence of the IF domain. The inserted domain decreased, however, the stability of the host domain in the transition region and decelerated its refolding reaction by about 10-fold. This originates from the interruption of the chain connectivity by the IF domain and its inherent instability. To monitor folding processes in this domain selectively, a Trp residue was introduced as fluorescent probe. Kinetic double-mixing experiments revealed that, in intact SlyD, the IF domain folds and unfolds about 1000-fold more rapidly than the FKBP domain, and that it is strongly stabilized when linked with the folded FKBP domain. The unfolding limbs of the kinetic chevrons of SlyD show a strong downward curvature. This deviation from linearity is not caused by a transition-state movement, as often assumed, but by the accumulation of a silent unfolding intermediate at high denaturant concentrations. In this kinetic intermediate, the FKBP domain is still folded, whereas the IF domain is already unfolded.     

Incipient forebrain boundaries traced by differential gene expression and fate mapping in the chick neural plate

We correlated available fate maps for the avian neural plate at stages HH4 and HH8 with the progress of local molecular specification, aiming to determine when the molecular specification maps of the primary longitudinal and transversal domains of the anterior forebrain agree with the fate mapped data. To this end, we examined selected gene expression patterns as they normally evolved in whole mounts and sections between HH4 and HH8 (or HH10/11 in some cases), performed novel fate-mapping experiments within the anterior forebrain at HH4 and examined the results at HH8, and correlated grafts with expression of selected gene markers. The data provided new details to the HH4 fate map, and disclosed some genes (e.g., Six3 and Ganf) whose expression domains initially are very extensive and subsequently retract rostralwards. Apart from anteroposterior dynamics, some genes soon became downregulated at the prospective forebrain floor plate, or allowed to identify an early roof plate domain (dorsoventral pattern). Peculiarities of the telencephalon (initial specification and differentiation of pallium versus subpallium) are contemplated. The basic anterior forebrain subdivisions seem to acquire correlated specification and fate mapping patterns around stage HH8.     

Structural and functional differences between KRIT1A and KRIT1B isoforms: a framework for understanding CCM pathogenesis

KRIT1 is a disease gene responsible for Cerebral Cavernous Malformations (CCM). It encodes for a protein containing distinct protein-protein interaction domains, including three NPXY/F motifs and a FERM domain. Previously, we isolated KRIT1B, an isoform characterized by the alternative splicing of the 15th coding exon and suspected to cause CCM when abnormally expressed.Combining homology modeling and docking methods of protein-structure and ligand binding prediction with the yeast two-hybrid assay of in vivo protein-protein interaction and cellular biology analyses we identified both structural and functional differences between KRIT1A and KRIT1B isoforms.We found that the 15th exon encodes for the distal β-sheet of the F3/PTB-like subdomain of KRIT1A FERM domain, demonstrating that KRIT1B is devoid of a functional PTB binding pocket. As major functional consequence, KRIT1B is unable to bind Rap1A, while the FERM domain of KRIT1A is even sufficient for this function. Furthermore, we found that a functional PTB subdomain enables the nucleocytoplasmic shuttling of KRIT1A, while its alteration confers a restricted cytoplasmic localization and a dominant negative role to KRIT1B. Importantly, we also demonstrated that KRIT1A, but not KRIT1B, may adopt a closed conformation through an intramolecular interaction involving the third NPXY/F motif at the N-terminus and the PTB subdomain of the FERM domain, and proposed a mechanism whereby an open/closed conformation switch regulates KRIT1A nuclear translocation and interaction with Rap1A in a mutually exclusive manner.As most mutations found in CCM patients affect the KRIT1 FERM domain, the new insights into the structure-function relationship of this domain may constitute a useful framework for understanding molecular mechanisms underlying CCM pathogenesis.     

应用微卫星标记进行大豆种质多样性和遗传变异性分析

微卫星标记又称ＳＳＲ标记是近年来发展的一种新型的分子标记可有效地进行基因型鉴定,系统谱分析并可估算材料间的遗传距离。用５对ＳＳＲ引物对１５份大豆材料进行扩增,共得到２１条多态性条带。每个ＳＳＲ座位的等侠基因数目为３～＾个,基因多样性范围为０．４３７～０．６６８,对这些材料进行了遗传距离分析。家系分析表明微卫星ＤＮＡ经过多世代的九分裂的后产生了突变,在ＲＩＬ Ｆ８代中有的个体在个别ＳＳＲ等基因的大小     

The energetics of phosphate binding to a protein complex

The heat of binding the serine protease, porcine pancreatic elastase, by the inhibitor, turkey ovomucoid third domain, is dependent on the presence of inorganic phosphate. This dependence is saturable and can be accurately modeled as the phosphate binding to a single site on the protease-inhibitor complex; thus, the elastase-ovomucoid system provides a unique opportunity to study phosphate-protein interactions. We have used isothermal titration calorimetry to investigate this binding, thereby providing one of the few complete thermodynamic characterizations of phosphate interacting with proteins. The binding is characterized by a small favorable deltaG degrees, a large unfavorable deltaH degrees, and a positive deltaCp, thermodynamics consistent with the release of water being linked to phosphate binding. These measurements provide insight into the binding of phosphotyrosine containing peptides to SH2 domains by suggesting the energetic consequences of binding phosphate free from other interactions.     

Notch signaling and Notch signaling modifiers

Originally discovered nearly a century ago, the Notch signaling pathway is critical for virtually all developmental programs and modulates an astounding variety of pathogenic processes. The DSL (Delta, Serrate, LAG-2 family) proteins have long been considered canonical activators of the core Notch pathway. More recently, a wide and expanding network of non-canonical extracellular factors has also been shown to modulate Notch signaling, conferring newly appreciated complexity to this evolutionarily conserved signal transduction system. Here, I review current concepts in Notch signaling, with a focus on work from the last decade elucidating novel extracellular proteins that up- or down-regulate signal potency.     

Paracrine regulation of growth factor signaling by shed leucine-rich repeats and immunoglobulin-like domains 1

Leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) is a recently discovered negative regulator of growth factor signaling. The LRIG1 integral membrane protein has been demonstrated to regulate various oncogenic receptor tyrosine kinases, including epidermal growth factor (EGF) receptor (EGFR), by cell-autonomous mechanisms. Here, we investigated whether LRIG1 ectodomains were shed, and if LRIG1 could regulate cell proliferation and EGF signaling in a paracrine manner. Cells constitutively shed LRIG1 ectodomains in vitro, and shedding was modulated by known regulators of metalloproteases, including the ADAM17 specific inhibitor TAPI-2. Furthermore, shedding was enhanced by ectopic expression of Adam17. LRIG1 ectodomains appeared to be shed in vivo, as well, as demonstrated by immunoblotting of mouse and human tissue lysates. Ectopic expression of LRIG1 in lymphocytes suppressed EGF signaling in co-cultured fibroblastoid cells, demonstrating that shed LRIG1 ectodomains can function in a paracrine fashion. Purified LRIG1 ectodomains suppressed EGF signaling without any apparent downregulation of EGFR levels. Taken together, the results show that the LRIG1 ectodomain can be proteolytically shed and can function as a non-cell-autonomous regulator of growth factor signaling. Thus, LRIG1 or its ectodomain could have therapeutic potential in the treatment of growth factor receptor-dependent cancers.     

Recognition of peptidoglycan and β-lactam antibiotics by the extracellular domain of the Ser/Thr protein kinase StkP from Streptococcus pneumoniae

The eukaryotic-type serine/threonine kinase StkP from Streptococcus pneumoniae is an important signal-transduction element that regulates the expression of numerous pneumococcal genes. We have expressed the extracellular C-terminal domain of StkP kinase (C-StkP), elaborated a three-dimensional structural model and performed a spectroscopical characterization of its structure and stability. Biophysical experiments show that C-StkP binds to synthetic samples of the cell wall peptidoglycan (PGN) and to β-lactam antibiotics, which mimic the terminal portions of the PGN stem peptide. This is the first experimental report on the recognition of a minimal PGN unit by a PASTA-containing kinase, suggesting that non-crosslinked PGN may act as a signal for StkP function and pointing to this protein as an interesting target for β-lactam antibiotics.     

Transgenic expression of the von Willebrand A domain of the BONZAI 1/COPINE 1 protein triggers a lesion-mimic phenotype in<Emphasis Type="Italic"> Arabidopsis</Emphasis>

The copines are a newly identified, widely distributed class of Ca²⁺-dependent, phospholipid-binding proteins that may be involved in cellular signaling. The copines have a characteristic domain structure: two C2 domains in the N-terminal region and a von Willebrand A (VWA) domain in the C-terminal region. Studies suggest that copines interact with target protein(s) via their VWA domain and recruit the proteins to a membrane location through the activity of the C2 domains. Arabidopsis thaliana (L.) Heynh. plants with loss-of-function mutations in the BONZAI 1/COPINE 1 (BON1/CPN1) gene display aberrant regulation of defense responses, including development of a lesion-mimic phenotype, an accelerated hypersensitive response, and increased resistance to a bacterial and an oomycetous pathogen. The phenotype of mutants in BON1/CPN1 is both humidity- and temperature-sensitive. In this study, we generated transgenic plants expressing either the VWA or the C2 portions of BON1/CPN1 in the wild-type Columbia-0 (Col-0) genetic background. Transgenic plants expressing the BON1/CPN1 C2 domain portion appeared like wild-type plants. However, transgenic plants expressing the BON1/CPN1 VWA domain exhibited a lesion-mimic phenotype that partially phenocopied bon1/cpn1 mutant plants. Our data suggest that BON1/CPN1 VWA domain fragments may interfere with the function of the full-length endogenous BON1/CPN1 protein, possibly by competing with the full-length BON1/CPN1 protein for association with target proteins normally bound to the full-length BON1/CPN1 protein.