Similarity of binding sites of human matrix metalloproteinases

Tissue components hydrolyzing matrix metalloproteinases (MMPs) exhibit a high sequence similarity (56-64% in catalytic domains) and yet a significant degree of functional specificity. The hexapeptide-binding sites of 24 known human MMPs were compared in terms of their force field interaction energies with five probes that are most frequently encountered in substrates and inhibitors. The probes moved along a grid enclosing partially flexible binding sites in rigid catalytic domains that were represented by published experimental structures and comparative models and new comparative models for nine most recently characterized MMPs. For individual MMPs, representative interaction energies were obtained as averages for all suitable experimental structures. Correlations of the representative energies for all MMP pairs were succinctly catalogued for individual probes, subsites, and correlation levels. Among the probes (neutral sp(3) carbon and sp(3) oxygen, positive sp(3) nitrogen and hydrogen, and negative carbonyl oxygen), the last probe is least distinctive. Similarities of subsites are decreasing as S1 ' > S2 > S3 ' > S1 approximately S3 > S2 '. Most interesting, occupancies of subsites in published structures of MMP-inhibitor complexes follow an almost parallel trend, alluding to overall low selectivity of known MMP inhibitors. Flexible subsite S1 ' that appears as the specificity pocket in rigid x-ray structures is actually very similar among individual MMPs. Several correlations indicated that MMPs 3, 8, and 12 have similar binding sites. Modeling results are corroborated with published experimental data on MMP inhibition and substrate specificities. The results provide numerous clues for development of specific inhibitors and substrates, as well as for selection of MMPs for testing that provides maximum information without redundant experiments.     

Similarity networks of protein binding sites

An increasing attention has been dedicated to the characterization of complex networks within the protein world. This work is reporting how we uncovered networked structures that reflected the structural similarities among protein binding sites. First, a 211 binding sites dataset has been compiled by removing the redundant proteins in the Protein Ligand Database (PLD) (http://www-mitchell.ch.cam.ac.uk/pld/). Using a clique detection algorithm we have performed all-against-all binding site comparisons among the 211 available ones. Within the set of nodes representing each binding site an edge was added whenever a pair of binding sites had a similarity higher than a threshold value. The generated similarity networks revealed that many nodes had few links and only few were highly connected, but due to the limited data available it was not possible to definitively prove a scale-free architecture. Within the same dataset, the binding site similarity networks were compared with the networks of sequence and fold similarity networks. In the protein world, indications were found that structure is better conserved than sequence, but on its own, sequence was better conserved than the subset of functional residues forming the binding site. Because a binding site is strongly linked with protein function, the identification of protein binding site similarity networks could accelerate the functional annotation of newly identified genes. In view of this we have discussed several potential applications of binding site similarity networks, such as the construction of novel binding site classification databases, as well as the implications for protein molecular design in general and computational chemogenomics in particular.     

NMR mapping of copper binding sites in alpha-synuclein

Copper binding to the Parkinson disease-linked protein alpha-synuclein (aS) has been shown to accelerate its oligomerization in vitro and may therefore play a role in aS-mediated pathology in vivo. We use NMR spectroscopy to identify a number of independent copper binding sites in both the lipid-binding N-terminal domain and the highly acidic C-terminal domain of aS. Most of the sites appear to involve negatively charged amino acid side chains, but binding is also observed to the sole histidine residue located at position 50 and to the N-terminal amino group. Both the N-terminal and the histidine sites, as well as the sites in the C-terminal tail, can also bind copper in the more highly structured conformation adopted by aS upon binding to detergent micelles or lipid vesicles. There is no evidence for the formation of any sites requiring long-range order in the protein.     

Biochemical characterization of the fibronectin binding sites for IgG

Plasma fibronectin (Fn) is a constituent of cryoglobulins and has been shown to interact with immune complexes. In a previous report we demonstrated that Fn specifically bound to IgG immobilized on a solid matrix. To localize and biochemically characterize the sites on the Fn molecule involved in this interaction, Fn was enzymatically cleaved with subtilisin and subjected to IgG affinity chromatography. Three major polypeptide fragments of 16 kDa, 22 kDa, and a triplet of 26- to 29-kDa bound IgG. They were localized to three separate regions of the molecule by Western blot analysis using antisera to specific regions of the Fn molecule, by amino acid sequencing, and by their previously described heparin binding affinities. The 22-kDa fragment interacted with IgG under physiologic conditions and it is localized at the N-terminal of the Fn molecule. The 16-kDa and 26- to 29-kDa fragments bound to IgG under conditions of lower ionic strength; the former commences at residue 588, carboxyl-terminal to the collagen binding region and the latter begins at residue 1597, carboxyl-terminal to the cell binding domain. The interaction of Fn with Ig has significant implications in host defense and also in immune complex disease where basement membrane Fn may sequester immune complexes from the circulation.     

Prolactin binding sites in human erythrocytes and lymphocytes

Specific binding sites for prolactin (PRL) have been studied in human peripheral lymphocytes and erythrocytes of normal adult volunteers and of term cord bloods. In erythrocytes from healthy adult subjects of both sexes a very low specific binding of 125I-human PRL was found (0.24%), whereas a higher binding was found in term cord blood (1.1%). The binding was hormone specific, the binding capacity was 2.6 fmol/4 X 10(9) cells and the Kd was 3.4 X 10(-10) M. In lymphocytes of both adults and term cord bloods an evident specific binding was observed (male adults: 1.6%; female adults: 1.7%; cord blood: 1.8%). The binding was specific for lactogenic hormones and the binding capacity was 3.7 fmol/2 X 10(6) cells and the Kd was 3.9 X 10(-10) M. The presence of specific binding sites for PRL on human erythrocytes and lymphocytes could be used to study PRL binding on blood cells of patients in different physiological or pathological situations.     

Prostaglandin binding sites in human polymorphonuclear neutrophils

Prostaglandin (PG) E2 (greater than or equal to 1.6 nM) and PGD2 (greater than or equal to 16 nM) inhibited polymorphonuclear neutrophil (PMN) degranulation responses to leukotriene (LT) B4 and platelet-activating factor (PAF) whereas PGF2 alpha was bioinactive. [3H]PGE2 and [3H]PGD2 bound to PMN and isolated, plasmalemma-enriched PMN membranes. Binding was time-dependent, specific, saturable, and reversible. Competitive studies indicated that the two PGs bound to distinctly different sites. PMN had high (Kd = 1 nM; Rt = 150/cell) and low (Kd = 100 nM; Rt = 5800/cell) affinity PGE2 binding sites. Only a single type of PGD2 binding site (Kd = 13 nM; Rt = 5100/cell) was detected. We conclude that PGE2 and PGD2 bind to their respective, plasmalemmal receptors to attenuate PMN function. The PGs may act as endogenous stop signals to limit the action of concurrently formed excitatory signals, eg., LTB4 and PAF.     

Serotonin binding sites of human blood platelets

The possible use of formaldehyde-fixed platelets to characterize and enumerate the specific receptor sites for 5-hydroxytryptamine was investigated. Equilibrium, pH-dependent capacity and specificity of 5-hydroxytryptamine binding by formaldehyde-fixed platelets were demonstrated. Analysis of binding data revealed two different sites: (i) high affinity with low capacity, and (ii) low affinity with high capacity. The results of binding studies using nonfixed control platelets were comparable with those of formaldehyde-fixed platelets. The versatility of formaldehyde fixation for studies of surface receptors was also shown by demonstrating nearly equal binding affinity for PGE₁ in control and formaldehyde-treated platelets. Our results indicate that formaldehyde fixation is a useful tool for the study of membrane receptor sites especially when active transport of the ligand such as serotonin is a problem.     

Inhaled anesthetic binding sites in human serum albumin

Previous evidence suggests multiple anesthetic binding sites on human serum albumin, but to date, we have only identified Trp-214 in an interdomain cleft as contributing to a binding site. We used a combination of site-directed mutagenesis, photoaffinity labeling, amide hydrogen exchange, and tryptophan fluorescence spectroscopy to evaluate the importance to binding of a large domain III cavity and compare it to binding character of the 214 interdomain cleft. The data show anesthetic binding in this domain III cavity of similar character to the interdomain cleft, but selectivity for different classes of anesthetics exists. Occupancy of these sites stabilizes the native conformation of human serum albumin. The features necessary for binding in the cleft appear to be fairly degenerate, but in addition to hydrophobicity, there is evidence for the importance of polarity. Finally, myristate isosterically competes with anesthetic binding in the domain III cavity and allosterically enhances anesthetic binding in the interdomain cleft.     

Hydrophobic binding sites on human interferon.

Human interferon binds to a omega-carboxpentyl-agarose column at low ionic strength (0.15 M NaCl) and is still retained when the ionic strength is raised (to 1.0 M NaCl). The binding can be reversed, however, by ethylene glycol, indicating a hydrophobic interaction. The binding of human interferon to omega-aminohexyl-agarose is weak, even at a low ionic strength, and is probably exclusively electrostatic. This disparate binding behavior may be caused by the presence of a positive charge, adjacent to the hydrophobic binding site, on human interferon. The interaction of human interferon with omega-carboxypentyl-agarose is quite selective, inasmuch as the majority of proteins present in interferon preparations pass through the column unretained. Hydrophobic chromatography of human interferon may thus be useful in its purification.     

Lectin binding sites in the human gallbladder and cystic duct

A battery of 18 fluorescein isothiocyanate (FITC) labeled lectins was used to histochemically define the features of epithelial cells in the body, neck and the cystic duct of the human gallbladder. Surface epithelium in all three anatomic locations reacted with all lectins, either diffusely or focally, except for lectin type I from Griffonia simplicifolia and type II from Ulex europaeus. No quantitative differences were noted except for the tendency of some lectins to bind more often to the neck and cystic duct epithelium rather than the body and vice versa. In the body the surface epithelium did not differ from cells lining the crypts. On the other hand, glands of the neck and the cystic duct were essentially indistinguishable one from another but differed from the overlying surface epithelium in so far as they reacted with some lectins which were unreactive with surface epithelium and vice versa. Considerable case to case variation in the expression of lectin binding sites in each of three anatomic areas was noted. No consistent differences were noticed between gallbladders removed for cholecystitis--cholelithiasis and those removed for other incidental reasons. We conclude that all epithelial cells in the cholecysto-biliary tract are rich on glyconjugates but the pattern of expression varies depending on the anatomical location and the influence of poorly understood individual determinants.     

Lectin binding sites in the human gallbladder and cystic duct

Summary A battery of 18 fluorescein isothiocyanate (FITC) labeled lectins was used to histochemically define the features of epithelial cells in the body, neck and the cystic duct of the human gallbladder. Surface epithelium in all three anatomic locations reacted with all lectins, either diffusely or focally, except for lectin type I from Griffonia simplicifolia and type II from Ulex europaeus. No quantitative differences were noted except for the tendency of some lectins to bind more often to the neck and cystic duct epithelium rather than the body and vice versa. In the body the surface epithelium did not differe from cells lining the crypts. On the other hand, glands of the neck and the cystic duct were essentially indistinguishable one from another but differed from the overlying surface epithelium in so far as they reacted with some lectins which were unreactive with surface epithelium and vice versa. Considerable case to case variation in the expression of lectin binding sites in each of three anatomic areas was noted. No consistent differences were noticed between gallbladders removed for cholecystitis — cholelithiasis and those removed for other incidental reasons. We conclude that all epithelial cells in the cholecysto-biliary tract are rich on glyconjugates but the pattern of expression varies depending on the anatomical location and the influence of poorly understood individual determinants.     

Hydrophobic binding sites of human liver alanine aminopeptidase

Fatty acids, alkyl amines, and amides of α-amino fatty acids inhibit human liver alanine aminopeptidase apparently by binding to residue binding site 1 of the active center, i.e., the N-terminal binding site. The pK_i values of the acids, amines, and amides increase until the overall chain length reaches eight carbons. The pK_i values are the same for members of the series with chain lengths longer than eight carbon atoms. Assuming an extended structure of the inhibitors, this site will accommodate amino acid side chains of not longer than 11.7 Å from the α-carbon to the end of the chain. Long chain amino acids inhibit by binding apparently at residue site 3. The pK_i values of dl-α-amino acids from α-aminobutyric acid to α-aminodecanoic acid increase with the addition of each methylene unit. Thus, site 3 will accommodate amino acid side chains which are at least 13.0 Å from the α-carbon to the end of the chain. Methanol and other organic solvents reversibly inhibit the binding of substrates at pH 6.9 without affecting the maximum rate of catalysis. At lower pH values, the maximum rate of catalysis is lowered. Sodium chloride also inhibits substrate hydrolysis at pH 6.9 but does not affect the maximum rate of catalysis. The pK_i values of fatty acids, alkyl amines, and amino acids are strongly decreased by methanol and slightly increased by sodium chloride. These data indicate that a major portion of the interactions of the enzyme with fatty acids, amines, and amino acids is of a hydrophobic nature.     

Characterization of serotonin binding sites on human platelets

A high affinity, saturable 3H-spiroperidol binding site was identified for the first time on the intact human platelet, with drug affinities comparable to the serotonin-2 (S-2) receptor in human frontal cortex. The site was characterized by a KD of 2.7 +/- 0.3nM and a Bmax of 1.4 +/- 0.2 pmoles/10(8) platelets. A 3H-serotonin binding site was also found, with a KD of 42 +/- 8 nM, which appeared to represent the serotonin uptake site. No 3H-serotonin binding site with features of the serotonin-1 (S-1) receptor in brain was found on the platelet. Assay of 3H-spiroperidol binding to platelets may serve as an easily applied model for studying S-2 receptor function in man, and its relationship to age, hormonal, drug, and disease effects.     

Identification of nucleoside transport binding sites in the human myocardium

The role of nucleoside transport in ischemia-reperfusion injury and arrhythmias has been well documented in various animal models using selective blockers. However, clinical application of nucleoside transport inhibitors remains to be demonstrated in humans. It is not known whether human heart has nucleoside transport similar to that of animals. The aim of this study is to pharmacologically identify the presence of nucleoside transport binding sites in the human myocardium compared to animals.Myocardial tissue was obtained from guinea pig left and right ventricle, canine left ventricle, human intraoperative right atrium and human cadaveric right atrium and right and left ventricles. Myocardial preparations were obtained from tissue samples after homogenized and a differential centrifugation.Equilibrium binding assays were performed using [³H]-p-nitrobenzylthioinosine (NBMPR) at room temperature in the presence or absence of non-radioactive NBMPR or other nucleoside transport blockers such as p-nitrobenzylthioguanosine dipyridamole, lidoflazine, papaverin, adenosine and doxorubcine. From saturation curves and inhibition kinetics, we determined the relative maximal binding (B_max) and dissociation constant (K_d) of [³H]-NBMPR binding of human myocardial preparations.Results demonstrated that the fresh human myocardial preparations have a specific binding site for NBMPR with a B_max of 283 ± 32 fmol/mg protein and K_d of 0.56 ± 0.12 nM. These values are lower than those obtained from guinea pigs (B_max = 1440 ± 187 fmol/mg protein and K_d = 0.21 ± 0.03 nM) and canine atrium (B_max 594 ± 73 fmol/mg protein, and K_d = 1.12 ± 0.22 nM).Displacement kinetics studies revealed the relative potencies (of certain unrelated drugs as follow: p-nitrobenzylthioguanosine > dipyridamole > lidoflazine > pavaverine > Diltazam > adenosine > doxyrubicin. It is concluded that human myocardium contains an active nucleoside transport site which may play a crucial role in post-ischemic reperfusion-mediated injury in a wide spectrum of ischemic syndromes.     

Properties of 3H-MPTP binding sites in human blood platelets

Our study demonstrates that 3H-1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (3H-MPTP) specifically binds to platelet membrane sites in humans. This specific, high affinity and saturable binding has properties similar to those of 3H-MPTP binding to rat and monkey brain, with a higher affinity. Deprenyl, a specific inhibitor of MAO type B enzyme, was the most potent drug in displacing 3H-MPTP from platelet binding sites. Platelets are considered a good model for central aminergic neurons and are very rich with MAO enzymatic activity, exclusively of type B. Our findings support previous evidence indicating a correspondence between 3H-MPTP binding sites and MAO-B enzyme. Moreover the presence of 3H-MPTP binding sites on human platelets suggests the use of this peripheral tissue as a simple model to study at least partially the mechanisms of neurotoxic action of MPTP.