scholarly journals Identification of a band-3 binding site near the N-terminus of erythrocyte membrane protein 4.2

1995 ◽  
Vol 309 (2) ◽  
pp. 677-681 ◽  
Author(s):  
A C Rybicki ◽  
S Musto ◽  
R S Schwartz
Keyword(s):  
Amino Acid ◽  
Membrane Protein ◽  
Binding Site ◽  
Human Erythrocyte ◽  
Basic Amino Acid ◽  
Band 3 ◽  
Amino Acid Residues ◽  
Protein 4.2 ◽  
Erythrocyte Plasma Membrane

Protein 4.2 (P4.2) is a major component of the erythrocyte plasma membrane accounting for approx. 5% of total membrane protein. The major membrane binding site for P4.2 is contained within the cytoplasmic domain of band 3 (cdb3), although the precise location of the cdb3 binding site is not known. To identify the cdb3 binding site, we used synthetic P4.2 peptides (15-mers) that spanned the entire 721-amino-acid large isoform of P4.2, and determined the binding of these peptides to cdb3 in an in vitro binding assay. One peptide, P8 (L61FVRRGQPFTIILYF), bound strongly to cdb3 and four others bound less strongly (P22, L271LNKRRGSVPILRQW; P27, G346EGQRGRIWIFQTST; P41, L556WRKKLHLTLSANLE; P48, I661HRERSYRFRSVWPE). These peptides have in common a cluster of two or three basic amino acid residues (arginine or lysine), in a region without nearby acidic residues. Cdb3 bound saturably to P8 with a Kd of 0.16 microM and a capacity of 0.56 mol of cdb3 monomer/mol of P8. Use of overlapping synthetic peptides further defined the cdb3 site as being contained within V63RRGQPFTIILYF. Replacement of R64R with R64G, G64R or G64G almost completely abolished cdb3 binding, suggesting that R64R is essential for cdb3 binding. P8 competitively inhibited binding of purified human erythrocyte P4.2 to cdb3. In blot overlay assays, cdb3 bound to a 23 kDa N-terminal P4.2 tryptic peptide containing V63RRGQPFTIILYF but not to other P4.2 tryptic peptides lacking this site. The V63RRGQPFTIILYF site is highly conserved in mouse and human erythrocyte P4.2 as well as between P4.2 and transglutaminase proteins, which are evolutionarily related to P4.2.

scholarly journals Mapping of a palmitoylatable band 3-binding domain of human erythrocyte membrane protein 4.2

1999 ◽  
Vol 340 (2) ◽  
pp. 505-512 ◽  
Author(s):  
Raja BHATTACHARYYA ◽  
Amit K. DAS ◽  
Prasun K. MOITRA ◽  
Biswajit PAL ◽  
Indranil MANDAL ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Erythrocyte Membrane ◽  
Human Erythrocyte ◽  
Band 3 ◽  
Amino Acid Residues ◽  
Fusion Peptides ◽  
Human Erythrocyte Membrane ◽  
Erythrocyte Membrane Protein ◽  
Protein 4.2 ◽  
Positive Modulator

Evidence accumulated over the years suggests that human erythrocyte membrane protein 4.2 is one of the proteins involved in strengthening the cytoskeleton-membrane interactions in the red blood cell. Deficiency of protein 4.2 is linked with a variety of hereditary haemolytic anaemia. However, the interactions of protein 4.2 with other proteins of the erythrocyte membrane remain poorly understood. The major membrane-binding site for protein 4.2 resides on the cytoplasmic domain of band 3 (CDB3). In order to carry out an initial characterization of its interaction with the CDB3, protein 4.2 was subjected to proteolytic cleavage and gel renaturation assay, and the 23-kDa N-terminal domain was found to interact with band 3. This domain contained two putative palmitoylatable cysteine residues, of which cysteine 203 was identified as the palmitoylatable cysteine. Recombinant glutathione S-transferase-fusion peptides derived from this domain were characterized with respect to their ability to interact with the CDB3. Whereas these studies do not rule out the involvement of other subsites on protein 4.2 in interaction with the CDB3, the evidence suggests that the region encompassing amino acid residues 187-211 is one of the domains critical for the protein 4.2-CDB3 interaction. This is also the first demonstration that palmitoylation serves as a positive modulator of this interaction.

scholarly journals Basic amino acid residue cluster within nuclear targeting sequence motif is essential for cytoplasmic plectin-vimentin network junctions.

1996 ◽  
Vol 134 (6) ◽  
pp. 1455-1467 ◽  
Author(s):  
B Nikolic ◽  
E Mac Nulty ◽  
B Mir ◽  
G Wiche
Keyword(s):  
Amino Acid ◽  
Intermediate Filaments ◽  
Basic Amino Acid ◽  
Site Directed Mutagenesis ◽  
Sequence Motif ◽  
Amino Acid Residues ◽  
Nuclear Targeting ◽  
Mutant Proteins ◽  
Carboxy Terminal

We have generated a series of plectin deletion and mutagenized cDNA constructs to dissect the functional sequences that mediate plectin's interaction with intermediate filament (IF) networks, and scored their ability to coalign or disrupt intermediate filaments when ectopically expressed in rat kangaroo PtK2 cells. We show that a stretch of approximately 50 amino acid residues within plectin's carboxy-terminal repeat 5 domain serves as a unique binding site for both vimentin and cytokeratin IF networks of PtK2 cells. Part of the IF-binding domain was found to constitute a functional nuclear localization signal (NLS) motif, as demonstrated by nuclear import of cytoplasmic proteins linked to this sequence. Site directed mutagenesis revealed a specific cluster of four basic amino acid residues (arg4277-arg4280) residing within the NLS sequence motif to be essential for IF binding. When mutant proteins corresponding to those expressed in PtK2 cells were expressed in bacteria and purified proteins subjected to a sensitive quantitative overlay binding assay using Eu3+-labeled vimentin, the relative binding capacities of mutant proteins measured were fully consistent with the mutant's phenotypes observed in living cells. Using recombinant proteins we also show by negative staining and rotary shadowing electron microscopy that in vitro assembled vimentin intermediate filaments become packed into dense aggregates upon incubation with plectin repeat 5 domain, in contrast to repeat 4 domain or a mutated repeat 5 domain.

scholarly journals The penicillin binding protein 1A of Helicobacter pylori, its amoxicillin binding site and access routes

Gut Pathogens ◽  
2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Bahareh Attaran ◽  
Najmeh Salehi ◽  
Bahareh Ghadiri ◽  
Maryam Esmaeili ◽  
Shadi Kalateh ◽  
...  
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
3D Structure ◽  
Resistance Mechanisms ◽  
Drug Binding ◽  
In Vitro Assays ◽  
Amino Acid Residues ◽  
Clinical Strains ◽  
H Pylori

Abstract Background Amoxicillin-resistant H. pylori strains are increasing worldwide. To explore the potential resistance mechanisms involved, the 3D structure modeling and access tunnel prediction for penicillin-binding proteins (PBP1A) was performed, based on the Streptococcus pneumoniae, PBP 3D structure. Molecular covalent docking was used to determine the interactions between amoxicillin (AMX) and PBP1A. Results The AMX-Ser368 covalent complex interacts with the binding site residues (Gly367, Ala369, ILE370, Lys371, Tyr416, Ser433, Thr541, Thr556, Gly557, Thr558, and Asn560) of PBP1A, non-covalently. Six tunnel-like structures, accessing the PBP1A binding site, were characterized, using the CAVER algorithm. Tunnel-1 was the ultimate access route, leading to the drug catalytic binding residue (Ser368). This tunnel comprises of eighteen amino acid residues, 8 of which are shared with the drug binding site. Subsequently, to screen the presence of PBP1A mutations, in the binding site and tunnel residues, in our clinical strains, in vitro assays were performed. H. pylori strains, isolated under gastroscopy, underwent AMX susceptibility testing by E-test. Of the 100 clinical strains tested, 4 were AMX-resistant. The transpeptidase domain of the pbp1a gene of these resistant, plus 10 randomly selected AMX-susceptible strains, were amplified and sequenced. Of the amino acids lining the tunnel-1 and binding site residues, three (Ser414Arg, Val469Met and Thr556Ser) substitutions, were detected in 2 of the 4 resistant and none of the sequenced susceptible strains, respectively. Conclusions We hypothesize that mutations in amino acid residues lining the binding site and/or tunnel-1, resulting in conformational/spatial changes, may block drug binding to PBP1A and cause AMX resistance.

scholarly journals Mapping of a palmitoylatable band 3-binding domain of human erythrocyte membrane protein 4.2

1999 ◽  
Vol 340 (2) ◽  
pp. 505 ◽  
Author(s):  
Raja BHATTACHARYYA ◽  
Amit K. DAS ◽  
Prasun K. MOITRA ◽  
Biswajit PAL ◽  
Indranil MANDAL ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Erythrocyte Membrane ◽  
Human Erythrocyte ◽  
Band 3 ◽  
Binding Domain ◽  
Human Erythrocyte Membrane ◽  
Erythrocyte Membrane Protein ◽  
Protein 4.2

A Cluster of Basic Amino Acid Residues in the γ370−381 Sequence of Fibrinogen Comprises a Binding Site for Platelet Integrin αIIbβ3(Glycoprotein IIb/IIIa)†

Biochemistry ◽  
2005 ◽  
Vol 44 (51) ◽  
pp. 16920-16930 ◽  
Author(s):  
Nataly P. Podolnikova ◽  
Oleg V. Gorkun ◽  
Ralph M. Loreth ◽  
Vivien C. Yee ◽  
Susan T. Lord ◽  
...  
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
Basic Amino Acid ◽  
Amino Acid Residues ◽  
Integrin Αiibβ3

scholarly journals Discovery of New Classes of Glycine transporter 2 (GlyT2) Inhibitors and Study of GlyT2 Selectivity by Combination of Novel Structural Based Virtual Screening Approach and Free Energy Perturbation (FEP+) Calculations

2019 ◽  
Author(s):  
Filip Fratev ◽  
Manuel Miranda-Arango ◽  
Elvia Padilla ◽  
Suman Sirimulla
Keyword(s):  
Chronic Pain ◽  
Free Energy ◽  
Amino Acid ◽  
Virtual Screening ◽  
Basic Amino Acid ◽  
Free Energy Perturbation ◽  
Amino Acid Residues ◽  
Strong Inhibitor ◽  
Energy Perturbation

In recent years, the mammalian GlyT2 transporter have emerged as a promising target for the development of anti-chronic pain agents. In our current work, we discovered a new set of promising hits that inhibit the glycine transport at nano and micromolar activity and have excellent selectivity over GlyT1 (as shown by in vitro studies), using a newly designed virtual screening (VS) protocol that combines a structure-based pharmacophore and docking screens. Furthermore, the free energy perturbation (FEP+ protocol) calculations and molecular dynamics (MD) studies revealed the GlyT2 amino acid residues critical for the binding and selectivity of both Glycine and our Lead1 compound. The FEP+ results well-matched available literature mutational data proving the quality of generated GlyT2 structure. Based on these calculations we propose that Lead1 may also be a strong inhibitor of the neutral and basic amino acid transporter B (0+) (SLC6A14). Thus, the subsequent lead optimization and characterization of refined compounds may lead to both chronic pain and pancreatic cancer agents addressing an unmet and challenging clinical needs.

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