Identification of functionally important regions of a haemoglobin receptor from Neisseria meningitidis

The HmbR outer-membrane receptor enables Neisseria meningitidis to use haemoglobin (Hb) as a source of iron. This protein functions by binding Hb, removing haem from it, and releasing the haem into the periplasm. Functionally important HmbR receptor domains were discerned using a series of HmbR deletions and site-directed mutations. Mutations exhibiting similar defective phenotypes in N. meningitidis fell into two groups. The first group of mutations affected Hb binding and were located in putative extracellular loops (L) L2 (amino acid residues (aa) 192–230) and L3 (aa 254–284). The second group of mutations resulted in a failure to utilize Hb but proficiency in Hb binding was retained. These mutations localized to the putative extracellular loops L6 (aa 420–462) and L7 (aa 486–516). A highly conserved protein motif found in all haem/Hb receptors, within putative extracellular loop L7 of HmbR, is essential for Hb utilization but not required for Hb binding. This finding suggests a mechanistic involvement of this motif in haem removal from Hb. In addition, an amino-terminal deletion in the putative cork-like domain of HmbR affected Hb usage but not Hb binding. This result supports a role of the cork domain in utilization steps that are subsequent to Hb binding.

Download Full-text

Relief of autoinhibition of the electrogenic Na-HCO3 cotransporter NBCe1-B: role of IRBIT vs. amino-terminal truncation

AJP Cell Physiology ◽

10.1152/ajpcell.00352.2011 ◽

2012 ◽

Vol 302 (3) ◽

pp. C518-C526 ◽

Cited By ~ 47

Author(s):

Seong-Ki Lee ◽

Walter F. Boron ◽

Mark D. Parker

Keyword(s):

Amino Acid ◽

Relative Magnitude ◽

Amino Acid Residues ◽

Wild Type ◽

Amino Terminal ◽

Sodium Bicarbonate Cotransporter ◽

Electrode Voltage ◽

Binding Determinants ◽

The Relationship

Two maneuvers known to stimulate electrogenic sodium bicarbonate cotransporter 1 (NBCe1) activity are 1) deletion from the cytosolic amino-terminus (Nt) of NBCe1-C of an 87-amino acid sequence that contains an autoinhibitory domain (AID); and 2) binding of the protein IRBIT to elements within the same 87-amino acid module in a different variant, NBCe1-B. Helpful to understanding the relationship between these two phenomena would be an appreciation of the relative magnitude of stimulation caused by each maneuver for the same NBCe1 variant. In the present study, we performed two-electrode voltage-clamp on Xenopus oocytes expressing human NBCe1-B constructs, with and without human IRBIT constructs. We find that removal of the AID stimulates NBCe1-B to the same extent as coexpression of wild-type IRBIT. The potency of wild-type IRBIT apparently is reduced by the action of endogenous oocyte protein phosphatases: a mutant IRBIT that cannot be influenced by the action of protein phosphatase-1 stimulates NBCe1-B to an extent 50% greater than can be achieved by removal of the NBCe1-B AID. Thus the stimulatory effect of IRBIT cannot be explained solely by masking of autoinhibitory determinants within the AID. Finally, we find that an NBCe1-B construct that lacks amino acid residues 2–16 of the Nt is fully autoinhibited, but cannot be stimulated by IRBIT, indicating that autoinhibitory and IRBIT-binding determinants within the cytosolic Nt are not identical.

Download Full-text

The arrangement and role of some of the amino acid residues in the beta-ketoacyl synthetase site of chicken liver fatty acid synthetase.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)44201-3 ◽

1983 ◽

Vol 258 (20) ◽

pp. 12482-12486

Author(s):

J K Stoops ◽

S J Henry ◽

S J Wakil

Keyword(s):

Fatty Acid ◽

Amino Acid ◽

Fatty Acid Synthetase ◽

Chicken Liver ◽

Amino Acid Residues ◽

Liver Fatty Acid

Download Full-text

A Factor VIII Neutralizing Monoclonal Antibody and a Human Inhibitor Alloantibody Recognizing Epitopes in the C2 Domain Inhibit Factor VIII Binding to von Willebrand Factor and to Phosphatidylserine

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651588 ◽

1993 ◽

Vol 69 (03) ◽

pp. 240-246 ◽

Cited By ~ 140

Author(s):

Midori Shima ◽

Dorothea Scandella ◽

Akira Yoshioka ◽

Hiroaki Nakai ◽

Ichiro Tanaka ◽

...

Keyword(s):

Monoclonal Antibody ◽

Amino Acid ◽

Factor Viii ◽

Von Willebrand Factor ◽

Light Chain ◽

Amino Acid Residues ◽

Amino Terminal ◽

Neutralizing Monoclonal Antibody ◽

Von Willebrand ◽

Willebrand Factor

SummaryA neutralizing monoclonal antibody, NMC-VIII/5, recognizing the 72 kDa thrombin-proteolytic fragment of factor VIII light chain was obtained. Binding of the antibody to immobilized factor VIII (FVIII) was completely blocked by a light chain-specific human alloantibody, TK, which inhibits FVIII activity. Immunoblotting analysis with a panel of recombinant protein fragments of the C2 domain deleted from the amino-terminal or the carboxy-terminal ends demonstrated binding of NMC-VIII/5 to an epitope located between amino acid residues 2170 and 2327. On the other hand, the epitope of the inhibitor alloantibody, TK, was localized to 64 amino acid residues from 2248 to 2312 using the same recombinant fragments. NMC-VIII/5 and TK inhibited FVIII binding to immobilized von Willebrand factor (vWF). The IC50 of NMC-VIII/5 for the inhibition of binding to vWF was 0.23 μg/ml for IgG and 0.2 μg/ml for F(ab)'2. This concentration was 100-fold lower than that of a monoclonal antibody NMC-VIII/10 which recognizes the amino acid residues 1675 to 1684 within the amino-terminal portion of the light chain. The IC50 of TK was 11 μg/ml by IgG and 6.3 μg/ml by F(ab)'2. Furthermore, NMC-VIII/5 and TK also inhibited FVIII binding to immobilized phosphatidylserine. The IC50 for inhibition of phospholipid binding of NMC-VIII/5 and TK (anti-FVIII inhibitor titer of 300 Bethesda units/mg of IgG) was 10 μg/ml.

Download Full-text

A major factor VIII binding domain resides within the amino-terminal 272 amino acid residues of von Willebrand factor.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)47430-3 ◽

1987 ◽

Vol 262 (18) ◽

pp. 8443-8446 ◽

Cited By ~ 9

Author(s):

P A Foster ◽

C A Fulcher ◽

T Marti ◽

K Titani ◽

T S Zimmerman

Keyword(s):

Amino Acid ◽

Factor Viii ◽

Von Willebrand Factor ◽

Binding Domain ◽

Amino Acid Residues ◽

Amino Terminal ◽

Von Willebrand ◽

Willebrand Factor

Download Full-text

Chemical Modification of Lysozyme, Glucose 6-Phosphate Dehydrogenase, and Bovine Eye Lens Proteins Induced by Peroxyl Radicals: Role of Oxidizable Amino Acid Residues

Chemical Research in Toxicology ◽

10.1021/tx300372t ◽

2013 ◽

Vol 26 (1) ◽

pp. 67-77 ◽

Cited By ~ 28

Author(s):

Andrea Arenas ◽

Camilo López-Alarcón ◽

Marcelo Kogan ◽

Eduardo Lissi ◽

Michael J. Davies ◽

...

Keyword(s):

Amino Acid ◽

Chemical Modification ◽

Peroxyl Radicals ◽

Eye Lens ◽

Amino Acid Residues ◽

Lens Proteins ◽

Glucose 6 Phosphate Dehydrogenase ◽

Eye Lens Proteins

Download Full-text

Deletion of the Actin-Associated Tropomyosin Tpm3 Leads to Reduced Cell Complexity in Cultured Hippocampal Neurons—New Insights into the Role of the C-Terminal Region of Tpm3.1

Cells ◽

10.3390/cells10030715 ◽

2021 ◽

Vol 10 (3) ◽

pp. 715

Author(s):

Tamara Tomanić ◽

Claire Martin ◽

Holly Stefen ◽

Esmeralda Parić ◽

Peter Gunning ◽

...

Keyword(s):

Amino Acid ◽

Hippocampal Neurons ◽

Molecular Mechanisms ◽

Growth Cones ◽

Amino Acid Residues ◽

Terminal Amino Acid ◽

C Terminus ◽

Primary Mouse ◽

Terminal Amino

Tropomyosins (Tpms) have been described as master regulators of actin, with Tpm3 products shown to be involved in early developmental processes, and the Tpm3 isoform Tpm3.1 controlling changes in the size of neuronal growth cones and neurite growth. Here, we used primary mouse hippocampal neurons of C57/Bl6 wild type and Bl6Tpm3flox transgenic mice to carry out morphometric analyses in response to the absence of Tpm3 products, as well as to investigate the effect of C-terminal truncation on the ability of Tpm3.1 to modulate neuronal morphogenesis. We found that the knock-out of Tpm3 leads to decreased neurite length and complexity, and that the deletion of two amino acid residues at the C-terminus of Tpm3.1 leads to more detrimental changes in neurite morphology than the deletion of six amino acid residues. We also found that Tpm3.1 that lacks the 6 C-terminal amino acid residues does not associate with stress fibres, does not segregate to the tips of neurites, and does not impact the amount of the filamentous actin pool at the axonal growth cones, as opposed to Tpm3.1, which lacks the two C-terminal amino acid residues. Our study provides further insight into the role of both Tpm3 products and the C-terminus of Tpm3.1, and it forms the basis for future studies that aim to identify the molecular mechanisms underlying Tpm3.1 targeting to different subcellular compartments.

Download Full-text

Role of amino acid residues at the interface of α52asparginyl-N-glycosyl chain of human choriogonadotropin

Molecular and Cellular Endocrinology ◽

10.1016/s0303-7207(99)00026-x ◽

1999 ◽

Vol 150 (1-2) ◽

pp. 47-56 ◽

Cited By ~ 1

Author(s):

Kui Shao ◽

Sathyamangalam V. Balasubramanian ◽

Om P. Bahl

Keyword(s):

Amino Acid ◽

Amino Acid Residues ◽

Human Choriogonadotropin

Download Full-text

The functional dyad of scorpion toxin Pi1 is not itself a prerequisite for toxin binding to the voltage-gated Kv1.2 potassium channels

Biochemical Journal ◽

10.1042/bj20030115 ◽

2004 ◽

Vol 377 (1) ◽

pp. 25-36 ◽

Cited By ~ 50

Author(s):

Stéphanie MOUHAT ◽

Amor MOSBAH ◽

Violeta VISAN ◽

Heike WULFF ◽

Muriel DELEPIERRE ◽

...

Keyword(s):

Amino Acid ◽

Scorpion Toxin ◽

Xenopus Laevis Oocytes ◽

Amino Acid Residues ◽

Voltage Gated ◽

Toxin Binding ◽

Ic50 Values

Pi1 is a 35-residue scorpion toxin cross-linked by four disulphide bridges that acts potently on both small-conductance Ca2+-activated (SK) and voltage-gated (Kv) K+ channel subtypes. Two approaches were used to investigate the relative contribution of the Pi1 functional dyad (Tyr-33 and Lys-24) to the toxin action: (i) the chemical synthesis of a [A24,A33]-Pi1 analogue, lacking the functional dyad, and (ii) the production of a Pi1 analogue that is phosphorylated on Tyr-33 (P-Pi1). According to molecular modelling, this phosphorylation is expected to selectively impact the two amino acid residues belonging to the functional dyad without altering the nature and three-dimensional positioning of other residues. P-Pi1 was directly produced by peptide synthesis to rule out any possibility of trace contamination by the unphosphorylated product. Both Pi1 analogues were compared with synthetic Pi1 for bioactivity. In vivo, [A24,A33]-Pi1 and P-Pi1 are lethal by intracerebroventricular injection in mice (LD50 values of 100 and 40 µg/mouse, respectively). In vitro, [A24,A33]-Pi1 and P-Pi1 compete with 125I-apamin for binding to SK channels of rat brain synaptosomes (IC50 values of 30 and 10 nM, respectively) and block rat voltage-gated Kv1.2 channels expressed in Xenopus laevis oocytes (IC50 values of 22 µM and 75 nM, respectively), whereas they are inactive on Kv1.1 or Kv1.3 channels at micromolar concentrations. Therefore, although both analogues are less active than Pi1 both in vivo and in vitro, the integrity of the Pi1 functional dyad does not appear to be a prerequisite for the recognition and binding of the toxin to the Kv1.2 channels, thereby highlighting the crucial role of other toxin residues with regard to Pi1 action on these channels. The computed simulations detailing the docking of Pi1 peptides on to the Kv1.2 channels support an unexpected key role of specific basic amino acid residues, which form a basic ring (Arg-5, Arg-12, Arg-28 and Lys-31 residues), in toxin binding.

Download Full-text