Abstract 52: The Role of Kindlin-2 in Integrin Activation Depends on a Short C-Terminal Region

2012 ◽  
Vol 32 (suppl_1) ◽  
Author(s):  
Jamila Hirbawi ◽  
Kamila Bledzka ◽  
Yan Qing Ma ◽  
Jun Qin ◽  
Edward F Plow
Keyword(s):  
Amino Acids ◽  
Point Mutation ◽  
Single Point ◽  
Terminal Region ◽  
Membrane Receptors ◽  
Single Point Mutation ◽  
Loss Of Function ◽  
Integrin Activation ◽  
C Terminus

Integrins are heterodimeric cell membrane receptors that regulate cell adhesion, migration, and survival. The kindlins are known to be key regulators of integrin activation, the transition from a low affinity, default state to a high affinity state for ligand. This function depends on their binding, together with talin, to the cytoplasmic tails (CT) of the β subunit of integrins. Kindlins are FERM domain containing proteins, and it is its F3 (PTB) subdomain of the FERM that is the primary binding site for integrin β CT. At its very C-terminus, beyond the F3, is a short extension of 21 amino acids, K2 660-680, and we have focused on the role of this region in the co-activator function of kindlin-2 (K2). For this analysis, we performed PAC-1 (antibody to detect activated αIIbβ3 integrin) binding assays in CHO cells stably expressing integrin α IIb β 3 that were transiently transfected with talin head domain and K2 mutants. Expression levels of all proteins were verified to be similar by western blotting and FACS. Truncation of K2 at residue 660 essentially eliminated the co-activator function of K2. Deletion of smaller segments also reduced co-activator activity by 50% to 100%. Deletion of just the last two amino acids in the sequence, W 679 V 680 , resulted in a 50% reduction in co-activator activity and a single point mutation of Y 673 A also led to a 50% loss of function. A combination mutant consisting of the W 679 V 680 deletion and the Y 673 point mutation resulted in 100% loss of kindlin-2 co-activator activity. Pull-down experiments performed using GST tagged β 3 CT and CHO lysates transfected with GFP-kindlin-2 forms suggested that the C-terminal deletion did not disrupt binding to β 3 CT. This observation was corroborated by surface plasmon resonance studies in which the binding of full-length K2 and K2Δ666C (Δ666) was compared, and their K D values for immobilized β3 CT were found to be essentially the same. Overall, these data establish an important and unanticipated role of the carboxy-terminal region of kindlin-2 in its integrin co-activator function that is not dependent of its binding to integrin.

scholarly journals Structural basis for a complex I mutation that blocks pathological ROS production

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Zhan Yin ◽  
Nils Burger ◽  
Duvaraka Kula-Alwar ◽  
Dunja Aksentijević ◽  
Hannah R. Bridges ◽  
...  
Keyword(s):  
Point Mutation ◽  
Complex I ◽  
Structural Changes ◽  
Ischemia Reperfusion ◽  
Single Point ◽  
Structural Basis ◽  
Single Point Mutation ◽  
Ros Production

AbstractMitochondrial complex I is central to the pathological reactive oxygen species (ROS) production that underlies cardiac ischemia–reperfusion (IR) injury. ND6-P25L mice are homoplasmic for a disease-causing mtDNA point mutation encoding the P25L substitution in the ND6 subunit of complex I. The cryo-EM structure of ND6-P25L complex I revealed subtle structural changes that facilitate rapid conversion to the “deactive” state, usually formed only after prolonged inactivity. Despite its tendency to adopt the “deactive” state, the mutant complex is fully active for NADH oxidation, but cannot generate ROS by reverse electron transfer (RET). ND6-P25L mitochondria function normally, except for their lack of RET ROS production, and ND6-P25L mice are protected against cardiac IR injury in vivo. Thus, this single point mutation in complex I, which does not affect oxidative phosphorylation but renders the complex unable to catalyse RET, demonstrates the pathological role of ROS production by RET during IR injury.

scholarly journals The Role of Chemically Modified DNA in Discrimination of Single-Point Mutation through Plasmon-Based Colorimetric Assays

2018 ◽  
Vol 1 (7) ◽  
pp. 3741-3746 ◽  
Author(s):  
María Sanromán-Iglesias ◽  
Charles H. Lawrie ◽  
Luis M. Liz-Marzán ◽  
Marek Grzelczak
Keyword(s):  
Point Mutation ◽  
Single Point ◽  
Single Point Mutation ◽  
Chemically Modified ◽  
Modified Dna ◽  
Colorimetric Assays

scholarly journals Assessing the capability ofin silicomutation protocols for predicting the finite temperature conformation of amino acids

2018 ◽  
Vol 20 (40) ◽  
pp. 25901-25909 ◽  
Author(s):  
Rodrigo Ochoa ◽  
Miguel A. Soler ◽  
Alessandro Laio ◽  
Pilar Cossio
Keyword(s):  
Molecular Dynamics ◽  
Amino Acids ◽  
Molecular Dynamics Simulations ◽  
Point Mutation ◽  
Finite Temperature ◽  
Single Point ◽  
Single Point Mutation ◽  
Equilibrium Configurations ◽  
Dynamics Simulations

Single-point mutation protocols based on backbone-dependent rotamer libraries show the best performance in predicting equilibrium configurations from molecular dynamics simulations.

scholarly journals High-Level Chromosomally Mediated Tetracycline Resistance in Neisseria gonorrhoeae Results from a Point Mutation in the rpsJ Gene Encoding Ribosomal Protein S10 in Combination with the mtrR and penB Resistance Determinants

2005 ◽  
Vol 49 (10) ◽  
pp. 4327-4334 ◽  
Author(s):  
Mei Hu ◽  
Sobhan Nandi ◽  
Christopher Davies ◽  
Robert A. Nicholas
Keyword(s):  
Amino Acids ◽  
Neisseria Gonorrhoeae ◽  
Ribosomal Protein ◽  
Point Mutation ◽  
Single Point ◽  
Ribosomal Subunit ◽  
Tetracycline Resistance ◽  
Saturation Mutagenesis ◽  
Single Point Mutation ◽  
High Level

ABSTRACT Neisseria gonorrhoeae becomes resistant to tetracycline by two major mechanisms: expression of a plasmid-encoded TetM protein and mutations in endogenous genes (chromosomally mediated resistance). Early studies by Sparling and colleagues (P. F. Sparling F. A. J. Sarubbi, and E. Blackman, J. Bacteriol. 124:740-749, 1975) demonstrated that three genes were involved in high-level chromosomally mediated tetracycline resistance (MIC of tetracycline ≥ 2 μg/ml): ery-2 (now referred to as mtrR), penB, and tet-2. While the identities of the first two genes are known, the tet-2 gene has not been identified. We cloned the tet-2 gene, which confers tetracycline resistance, from tetracycline-resistant clinical isolate N. gonorrhoeae FA6140 and show that resistance is due to a single point mutation (Val-57 to Met) in the rpsJ gene (rpsJ1) encoding ribosomal protein S10. Moreover, the identical mutation was found in six distinct tetracycline-resistant clinical isolates in which the MIC of tetracycline was ≥2 μg/ml. Site-saturation mutagenesis of the codon for Val-57 identified two other amino acids (Leu and Gln) that conferred identical levels of resistance as the Met-57 mutation. The mutation maps to the vertex of a loop in S10 that is near the aminoacyl-tRNA site in the structure of the 30S ribosomal subunit from Thermus thermophilus, and the residue equivalent to Val-57 in T. thermophilus S10, Lys-55, is within 8 to 9 Å of bound tetracycline. These data suggest that large noncharged amino acids alter the rRNA structure near the tetracycline-binding site, leading to a lower affinity of the antibiotic.

scholarly journals Importance of C-Terminal Knot Structure in Carbonic Anhydrase II (Part I): Role of C-Terminal Knot Forming GLN253 on Enzymatic Property of Human Carbinic II

1970 ◽  
Vol 14 ◽  
pp. 1-9
Author(s):  
Mohammad Taufiq Alam
Keyword(s):  
Amino Acid ◽  
Carbonic Anhydrase ◽  
Point Mutation ◽  
Amino Acid Residue ◽  
Active Site ◽  
Terminal Region ◽  
Carbonic Anhydrase Ii ◽  
C Terminus ◽  
Human Carbonic Anhydrase

In both, bovine and human carbonic anhydrase II, a conserved glutamine residue occupies the position in the middle of the knot, which is formed by intercrossing of C-terminal end with N-terminal region. Previous studies have indicated that C-terminus is not the part of an active site, but truncation of 7 amino acid residue in this region can have marked effects on stability of the enzyme (data not published). To gain further insight into the role of specific amino acid residue in C-terminal region, site directed mutagenesis was used to introduce point mutation. Substitution of glutamine with cysteine was chosen because the cysteine residue is less hydrophilic as compared with glutamine and thus, may disrupt the hydrophilic environment in this region. Result indicates that Gln253 located within the C-terminus knot topology plays a significant role in normal function of the enzyme. Thus, C-terminal region might mediate cooperativity between the central active site of the enzyme through proper formation of knot. Key words: Human carbonic anhydrase II; knot topology; point mutation J. bio-sci. 14: 1-9, 2006

scholarly journals A single point mutation converts a proton-pumping rhodopsin into a red-shifted, turn-on fluorescent sensor for chloride

2021 ◽  
Author(s):  
Jasmine N. Tutol ◽  
Jessica Lee ◽  
Hsichuan Chi ◽  
Farah N. Faizuddin ◽  
Sameera S. Abeyrathna ◽  
...  
Keyword(s):  
Point Mutation ◽  
Fluorescent Sensor ◽  
Single Point ◽  
Proton Pumping ◽  
Single Point Mutation ◽  
Turn On ◽  
Gloeobacter Violaceus

By utilizing laboratory-guided evolution, we have converted the fluorescent proton-pumping rhodopsin GR from Gloeobacter violaceus into GR1, a red-shifted, turn-on fluorescent sensor for chloride.

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