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PubMed1993

Molecular dynamics simulations of asialoglycoprotein receptor ligands.

Several recent studies have implicated carbohydrates in cell adhesion, inflammation, clearance of glycoproteins from blood circulation, embryonic development, and metastasis among others. Understanding the conformation of these carbohydrate recognition elements and their interaction at the molecular level is essential for the design of oligosaccharide inhibitors/drugs. Given the difficulty in solving carbohydrate structures by X-ray crystallography and since NMR experiments give only time-averaged conformation, molecular dynamics simulations are well suited to determine all the accessible conformations of oligosaccharides. Present communication reports the simulation of some of the oligosaccharide ligands of asialoglycoprotein receptor for 1 ns using Biosym's Insight II molecular modeling package on NCI-FCRDC's Y-MP 8D/8128 supercomputer. Results obtained from these simulations, in addition to explaining the observed differences in the binding affinities of these ligands to the asialoglycoprotein receptor, have led to a modified model for the recognition of the oligosaccharides by the receptor. Accordingly, only the two terminal galactose residues on the 1,3-arm of the triantennary oligosaccharide (GlcNAc2Man3 core of the N-linked oligosaccharides with N-acetyllactosamine in beta 1,2- and beta 1,4-linkages on the 1,3-linked core mannose) are primarily required for recognition, and the terminal galactose on the 1,6-arm (N-acetyllactosamine in beta 1,2-linkage on the 1,6-linked core mannose) provides additional binding energy. It has been shown that the oligosaccharides studied here have significant flexibility and the flexibility is more around the 1,3-linkage than the 1,6-linkage. The need for simulation for longer periods and with multiple initial conformations is also discussed in the present report.

Asialoglycoprotein Receptor↗
PubMed1997

Conformationally restrained melatonin analogues: synthesis, binding affinity for the melatonin receptor, evaluation of the biological activity, and molecular modeling study.

The design, synthesis, and biological profile of several indole melatonin analogues with a conformationally restricted C3 amidoethane side chain are presented. Examination of the accessible conformations of the melatonin side chain led us to explore some of its fully or partially restricted analogues, 2-12, the binding affinity values of which were utilized to gain further insight on the melatonin binding site. Two pharmacophoric models have been devised for melatonin and the active compounds by conformational analysis and superimposition performed using the DISCO program. In these models, the melatonin side chain can adopt a gauche/anti conformation out of the indole plane. Another contribution of this study regards the observation of a possible binding point interaction around the C2 position of the indole, as suggested by the remarkably increased binding affinity observed in the C2-substituted analogues 6 and 9 and especially in the more rigid analogue 5. The biological activity and the efficacy of the new compounds were tested by measuring the inhibition of the forskolin-stimulated cAMP accumulation and the GTP gamma S index. Both analyses demonstrated that all of the compounds were full agonists with the exception of 4 and 9, which showed a slight reduction in efficacy and would seem to be partial agonists.

Animals↗
PubMed2002

Newcastle disease virus HN protein alters the conformation of the F protein at cell surfaces.

Conformational changes in the Newcastle disease virus (NDV) fusion (F) protein during activation of fusion and the role of HN protein in these changes were characterized with a polyclonal antibody. This antibody was raised against a peptide with the sequence of the amino-terminal half of the F protein HR1 domain. This antibody immunoprecipitated both F(0) and F(1) forms of the fusion protein from infected and transfected cell extracts solubilized with detergent, and precipitation was unaffected by expression of the HN protein. In marked contrast, this antibody detected significant conformational differences in the F protein at cell surfaces, differences that depended upon HN protein expression. The antibody minimally detected the F protein, either cleaved or uncleaved, in the absence of HN protein expression. However, when coexpressed with HN protein, an uncleaved mutant F protein bound the anti-HR1 antibody, and this binding depended upon the coexpression of specifically the NDV HN protein. When the cleaved wild-type F protein was coexpressed with HN protein, the F protein bound anti-HR1 antibody poorly although significantly more than F protein expressed alone. Anti-HR1 antibody inhibited the fusion of R18 (octadecyl rhodamine B chloride)-labeled red blood cells to syncytia expressing HN and wild-type F proteins. This inhibition showed that fusion-competent F proteins present on surfaces of syncytia were capable of binding anti-HR1. Furthermore, only antibody which was added prior to red blood cell binding could inhibit fusion. These results suggest that the conformation of uncleaved cell surface F protein is affected by HN protein expression. Furthermore, the cleaved F protein, when coexpressed with HN protein and in a prefusion conformation, can bind anti-HR1 antibody, and the anti-HR1-accessible conformation exists prior to HN protein attachment to receptors on red blood cells.

Amino Acid Sequence↗
PubMed1993

The conformations of cyclic (1-->2)-beta-D-glucans: application of multidimensional clustering analysis to conformational data sets obtained by Metropolis Monte Carlo calculations.

Sets containing up to 1.3 x 10(6) energetically accessible conformations of linear (1-->2)-beta-D-glucan oligosaccharides were obtained by Metropolis Monte Carlo (MMC) calculations performed with the GEGOP (GEometry of GlycOProteins) program. Quantitative analyses of the data sets (which were expressed in terms of the glycosidic dihedral angle coordinates) were obtained by two different clustering methods: (i) the three-distance hierarchical clustering method (3-DM), published by Jure Zupan, and (ii) a nonhierarchical clustering method (Population-Density Projection, PDP) which, through a segmentation analysis of two-dimensional projections of the population-density surface, establishes a partitioning of conformational space into a set of "cluster regions", followed by a clustering step where each conformation of the data set is assigned to one of these regions. Computer programs (MCLUST and PDPCLUST) were developed to perform the 3-DM and PDP analyses, respectively. The two types of analysis provided very similar sets of conformational families (clusters), which could be expressed as combinations of distinct conformations of the glycosidic torsional angles (phi, psi) centered at (50 degrees, 10 degrees) for conformation A, (40 degrees, 160 degrees) for conformation B, (55 degrees, -160 degrees) for conformation B', and (170 degrees, 10 degrees) for conformation C. The analysis provided the populations of the families, along with relative rates for transitions between families. Examination of the frequencies of the A, B, and C glycosidic bond conformations with respect to their relative positions in the sequence revealed the tendency of the (1-->2)-beta-D-glucan to adopt conformational repeating structures of the general form [AnB], where n = 3 or 6. These repeating structures combine in an energetically cooperative fashion to give low-energy cyclic conformations having, for example C5 symmetry [AAAB]5 for the eicosamer, and C3 symmetry [AAAAAAB]3 for the heneicosamer.

Carbohydrate Conformation↗
PubMed2006

Quantifying polypeptide conformational space: sensitivity to conformation and ensemble definition.

Quantifying the density of conformations over phase space (the conformational distribution) is needed to model important macromolecular processes such as protein folding. In this work, we quantify the conformational distribution for a simple polypeptide (N-mer polyalanine) using the cumulative distribution function (CDF), which gives the probability that two randomly selected conformations are separated by less than a "conformational" distance and whose inverse gives conformation counts as a function of conformational radius. An important finding is that the conformation counts obtained by the CDF inverse depend critically on the assignment of a conformation's distance span and the ensemble (e.g., unfolded state model): varying ensemble and conformation definition (1 --> 2 A) varies the CDF-based conformation counts for Ala(50) from 10(11) to 10(69). In particular, relatively short molecular dynamics (MD) relaxation of Ala(50)'s random-walk ensemble reduces the number of conformers from 10(55) to 10(14) (using a 1 A root-mean-square-deviation radius conformation definition) pointing to potential disconnections in comparing the results from simplified models of unfolded proteins with those from all-atom MD simulations. Explicit waters are found to roughen the landscape considerably. Under some common conformation definitions, the results herein provide (i) an upper limit to the number of accessible conformations that compose unfolded states of proteins, (ii) the optimal clustering radius/conformation radius for counting conformations for a given energy and solvent model, (iii) a means of comparing various studies, and (iv) an assessment of the applicability of random search in protein folding.

Computer Simulation↗
PubMed2002

Arginine-349 and aspartate-373 of the Na(+)/dicarboxylate cotransporter are conformationally sensitive residues.

The conserved residues, Arg-349 and Asp-373, of the renal Na(+)/dicarboxylate cotransporter (NaDC-1) have been shown in our previous studies to affect substrate affinity and cation binding. In this study, amino acids surrounding Arg-349 and Asp-373 were individually mutated to cysteines and their sensitivity to methanethiosulfonate reagents (MTS) was tested. Only three of the 21 mutants were sensitive to MTS reagents: R349C, S372C, and D373C. The R349C mutant had reduced activity which was restored by chemical modification with MTSEA. The effect of MTSEA was only observed in the presence of sodium, indicating that Arg-349 is conformationally accessible. The succinate transport activity of the S372C mutant was stimulated by both MTSEA and MTSET. The D373C mutant was very sensitive to inhibition by MTSET (K(i) = 0.5 microM) in sodium buffer. The inhibition of D373C by MTSET was prevented by substrate, suggesting that the substrate-induced conformational change occludes the residue. We conclude that the accessibility of Arg-349 and Asp-373 is likely to change with the conformational states of the transport cycle.

Amino Acid Substitution↗
PubMed1995

A model for transmembrane helix with a cis-proline in the middle.

The presence of a higher percentage of Proline in the transmembrane helices of transport proteins indicates that they are involved in the function of these integral membrane proteins (IMPs). In many cases, the possible involvement of cis-trans isomerization in function/folding of IMPs has been suggested. The introduction of cis-Pro in an ideal alpha-helix results in a helix-turn-helix motif. A molecular dynamics (MD) simulation is carried out on the sequence ACE-(ALA)10-cis-Pro-(ALA)10-NME with ideal alpha-helical structure to investigate if and how a straight helix can accommodate a cis-Pro. The analysis of the conformations accessed during MD simulation showed that the residues near cis-Pro can adopt alternate conformations other than the right-handed helical conformation such that an almost straight helix is obtained. This may have implications in the involvement of cis-trans isomerization in folding and/or function of IMPs.

Membrane Proteins↗
PubMed2001

Conformations of an adenine bulge in a DNA octamer and its influence on DNA structure from molecular dynamics simulations.

Molecular dynamics simulations have been applied to the DNA octamer d(GCGCA-GAAC). d(GTTCGCGC), which has an adenine bulge at the center to determine the pathway for interconversion between the stacked and extended forms. These forms are known to be important in the molecular recognition of bulges. From a total of ~35 ns of simulation time with the most recent CHARMM27 force field a variety of distinct conformations and subconformations are found. Stacked and fully looped-out forms are in excellent agreement with experimental data from NMR and x-ray crystallography. Furthermore, in a number of conformations the bulge base associates with the minor groove to varying degrees. Transitions between many of the conformations are observed in the simulations and used to propose a complete transition pathway between the stacked and fully extended conformations. The effect on the surrounding DNA sequence is investigated and biological implications of the accessible conformational space and the suggested transition pathway are discussed, in particular for the interaction of the MS2 replicase operator RNA with its coat protein.

Adenine↗
PubMed1997

19-nor-10-azasteroids, a new class of steroid 5 alpha-reductase inhibitors. 2. X-ray structure, molecular modeling, conformational analysis of 19-nor-10-azasteroids and comparison with 4-azasteroids and 6-azasteroids.

19-Nor-10-azasteroids are a new class of 5 alpha-reductase inhibitors whose activity depends on the presence of the bridgehead N-10 atom conjugated with the 4-en-3-one moiety in the A ring. The X-ray structure of 19-nor-10-azasteroid 1 has been determined and it is compared with the X-ray structure of testosterone. A complete conformational analysis of these compounds has been performed, determining the number and energy of the possible conformers, as well as the molecular flexibility of the 10-azasteroidal skeleton. Thus, MM2* molecular mechanics calculations and AM1 semiempirical energy refinements revealed that 19-nor-10-azasteroids 1-3 have four possible conformations with very small energy differences and that they are very flexible molecules. The conformational analysis has been extended to testosterone (4), which also showed conformational flexibility, with three different conformations, and to 6-azasteroid 5 and 4-azasteroid 6, for which only two thermally accessible conformations have been found. Compared to 19-nor-10-azasteroids 1-3, azasteroids 5 and 6 appear to be more rigid structures. By a best fit analysis of all conformers of 1-5 with the global minimum of testosterone (4-I) it has been found that the lowest energy conformers of 1, 3, and 5 are very close to the structure of 4-I, and among the conformers of 2, the best similarity has been observed for the highest energy conformer 2-IV.

5-alpha Reductase Inhibitors↗
PubMed1992

Solution conformation of a pectin fragment disaccharide using molecular modelling and nuclear magnetic resonance.

In the present study, the conformational behaviour of methylated pectic disaccharide 4-O-alpha-D-galactopyranurosyl 1-O-methyl-alpha-D-galactopyranuronic 6,6'-dimethyl diester 1 has been completely characterized through combined n.m.r. and molecular modelling studies. The 1H-1H n.O.e. across the glycosidic bond was measured by both steady-state and transient 1D and 2D experiments. In parallel, the complete conformational analysis of the disaccharide has been achieved with the MM3 molecular mechanics method. The conformation of the pyranose ring is confirmed by the excellent agreement between the experimental and calculated intracyclic scalar coupling constants. The iso-energy contours displayed on the 'relaxed' map indicate an important flexibility about the glycosidic linkage. There is no significant influence of the methoxyl group on the conformational behaviour of the disaccharide. The theoretical n.m.r. data were calculated taking into account all the accessible conformations and using the averaging methods appropriate for slow internal motions. 3JC-H coupling constants were calculated using an equation suitable for C-O-C-H segments. The agreement between experimental and theoretical data is excellent. Within the potential energy surface calculated for the disaccharide, several conformers can be identified. When these conformations are extrapolated to a regular polymer structure, they generate pectins with right- and left-handed chirality along with a two-fold helix. These different types of helical structure are the result of small changes in conformation, without any drastic variation of the fibre repeat.

Carbohydrate Conformation↗
PubMed1985

[(Aminomethyl)arloxy]acetic acid esters. A new class of high-ceiling diuretics. 3. Variation in the bridge between the aromatic rings to complete mapping of the receptor.

Continued structural evaluation of the [(aminomethyl)aryloxy]acetic ester diuretics has produced a series of compounds in which the functional group that bridges the two aromatic rings has been varied. Diuretic screening of these analogues in rats indicates that the keto group can be effectively replaced with an ether or thio ether function with a slight increase in potency, whereas the methylene and sulfoxide linking groups lead to diminished saluretic potency. Replacement with either -SO2-, -COCO-, -CH2O-, -CONH- or direct bond results in a loss of activity. Although the series was designed according to QSAR criteria, the traditional linear free-energy properties of these compounds do not correlate with diuretic potency. However, conformational analysis of the series by potential energy calculations indicates that all active compounds have an accessible conformation that matches the bridge atom-carboxylate distance of the very potent dihydrobenzofuran analogue 56. Conformational calculations of several compounds in which the aminomethyl group was varied suggests that the active conformation is probably a low-energy conformation. Consideration of rotation about the bridge could not distinguish between two possible orientations of the aminomethyl ring in the active conformation. However, there is a quantitative negative linear correlation between diuretic potency and the protrusion into space of the group that bridges the two aromatic rings.

Animals↗
PubMed1992

Molecular determinants of mu receptor recognition for the fentanyl class of compounds.

We report here a theoretical study of a series of fentanyl analogs with a wide range of affinities and selectivities at the mu receptor, designed to identify and characterize the molecular determinants of mu receptor recognition. In this work, a complete conformational search combining nested rotations and molecular dynamic simulations has been made, leading to identification of accessible conformers for all analogs and to the selection of a candidate bioactive form. In addition, electronic properties have been calculated and examined as possible modulators of recognition at the mu receptor. The results of these studies have led to a distinct pharmacophore for interaction at the mu receptor for this class of compounds, with the piperidine ring in a chair conformation and the N-phenethyl and 4-phenylpropanamide substituents both equatorial. Moreover, four key moieties necessary for optimum receptor recognition and a postulated role for each of them in this recognition have been identified. These are (i) a protonated amine nitrogen, assumed to be involved in an initial electrostatic interaction with a negatively charged site on the receptor; (ii) a polar function capable of hydrogen-bonding with an electrophilic site; (iii) an aromatic ring involved in lipophilic interaction with a similar moiety; and (iv) a second aromatic ring, most probably involved in electron transfer interaction with the receptor. These requirements, taken together, form the basis of our proposed mechanism for mu receptor recognition. Not only is the presence of these components required for recognition, but specific steric relationships between them have been determined, implying the appropriate arrangement for interaction with complementary receptor sites. These steric parameters are pseudobond angles and one torsion angle that determine the relative spatial arrangement of these four moieties. They are the angles theta 1 and theta 3, defining the relative position of the protonated nitrogen and the polar function with each of the two aromatic rings, and the torsion angle eta 1, defining the orientation of the lone pair(s) on the polar proton-accepting function with respect to the lone pair on the piperidine nitrogen. This postulated mechanism of recognition provides a conceptual framework to understand why some compounds do and some do not recognize the mu receptor.

Fentanyl↗
PubMed2002

QSAR studies of the pyrethroid insecticides. Part 3. A putative pharmacophore derived using methodology based on molecular dynamics and hierarchical cluster analysis.

Previous studies of the conformational behaviour of a group of synthetic pyrethroid insecticides have been extended to a more structurally diverse set. This includes compounds with different backbones and differing stereochemistry, with both Types I and II biological activity. These compounds also encompass a large range of biological activities. A parameterisation of the CHARMM force field for these compounds has been performed and the extra parameters are reported. Conformational sampling, using molecular dynamics (MD), has been performed for each of the 41 active structures. The accessible conformations of each have been characterised by the values of the common torsion angles using hierarchichal cluster analysis (HCA). A further CA, based on the centroids derived from the conformational sampling, identified a conformation common to at least 39 of the 41 structures. The critical torsion angles of this conformation lie at the centre of the molecule about the ester linkage and are defining an extended conformation, which differs from the minimum energy conformation of deltamethrin used previously. This may represent a putative pharmacophore for kill. The methods used here improve significantly on those used previously. The CHARMM force field was parameterised for the compounds and an improved method of conformational sampling, based on centroid clustering, has also been used.

Allethrins↗
PubMed2004

How much pi-stacking do DNA termini seek? Solution structure of a self-complementary DNA hexamer with trimethoxystilbenes capping the terminal base pairs.

The exposed terminal base pairs of DNA duplexes are nonclassical binding sites for small molecules. Instead, small molecules usually prefer intercalation or minor groove binding. Here we report the solution structure of the DNA duplex (TMS-TGCGCA)(2), where TMS denotes trimethoxystilbene carboxamides that are 5'-tethered to the DNA. The stilbenes, for which intercalation is conformationally accessible, stack on the terminal T:A base pairs of an undisturbed B-form duplex. Two conformations, differing by the orientation of the stilbene relative to the terminal base pair, are observed, indicating that the flip rate is slow for the pi-stacked aromatic ring system. The trimethoxystilbene is known to greatly increase base pairing fidelity at the terminus. Here we show that it gauges the size of the T:A base pair by embracing the 2'-methylene group of the terminal dA residue of the unmodified terminus with its methoxy "arms", but that it does not engage the entire base pair in pi-stacking. Mismatched base pairs with their altered geometry will not allow for the same embracing interaction. On the basis of the current structure, a trimethoxychrysene carboxamide is proposed as a ligand with increased pi-stacking surface and possible applications as improved fidelity-enhancing element.

Base Pairing↗
PubMed1999

The solution structure of a superpotent B-chain-shortened single-replacement insulin analogue.

This paper reports on an insulin analogue with 12.5-fold receptor affinity, the highest increase observed for a single replacement, and on its solution structure, determined by NMR spectroscopy. The analogue is [D-AlaB26]des-(B27-B30)-tetrapeptide-insulin-B26-amide. C-terminal truncation of the B-chain by four (or five) residues is known not to affect the functional properties of insulin, provided the new carboxylate charge is neutralized. As opposed to the dramatic increase in receptor affinity caused by the substitution of D-Ala for the wild-type residue TyrB26 in the truncated molecule, this very substitution reduces it to only 18% of that of the wild-type hormone when the B-chain is present in full length. The insulin molecule in solution is visualized as an ensemble of conformers interrelated by a dynamic equilibrium. The question is whether the "active" conformation of the hormone, sought after in innumerable structure/function studies, is or is not included in the accessible conformational space, so that it could be adopted also in the absence of the receptor. If there were any chance for the active conformation, or at least a predisposed state to be populated to a detectable extent, this chance should be best in the case of a superpotent analogue. This was the motivation for the determination of the three-dimensional structure of [D-AlaB26]des-(B27-B30)-tetrapeptide-insulin-B26-amide. However, neither the NMR data nor CD spectroscopic comparison of a number of related analogues provided a clue concerning structural features predisposing insulin to high receptor affinity. After the present study it seems more likely than before that insulin will adopt its active conformation only when exposed to the force field of the receptor surface.

Circular Dichroism↗
PubMed2004

Experimental indication for the existence of multiple Trp rotamers in von Willebrand Factor A3 domain.

The first step in both normal haemostasis and arterial thrombosis is the interaction between collagen, von Willebrand factor (vWF), and glycoprotein Ib. The A3 domain of vWF forms the principal binding site for collagen type I and type III. Inhibition of the vWF-collagen interaction by an anti-human vWF monoclonal antibody (MoAb) 82D6A3 can be a potential way to prevent arterial thrombosis. Identification of the epitope of MoAb 82D6A3 showed recently that the consensus sequence SPWR obtained by phage display could adopt the conformation of the discontinuous epitope. Modelling showed that Trp982 in the vWF had to obtain a more solvent accessible conformation. We performed a detailed fluorescence study of Trp982 in the vWF A3. Using the method described by Hellings et al. (Biophys J 2003;85:1894-1902), we were able to identify two different low-energy Trp982 rotamers and to link them with their experimentally derived fluorescence lifetimes. Fluorescence anisotropy showed no interconversion in the nanosecond timescale between the two different rotameric states. With these experiments, we gather strong indications for the existence of an exposed rotamer conformation and a rotamer that corresponds to the one observed in the X-ray structure. These results strongly support the modeling work (Vanhoorelbeke et al., J Biol Chem 2003;278:37815-37821).

Binding Sites↗
PubMed2004

Targacept active conformation search: a new method for predicting the conformation of a ligand bound to its protein target.

Targacept active conformation search (TACS) is a novel variation of well-established three-dimensional quantitative structure--activity relationship methodologies that seeks to determine probable conformation(s) of ligands bound to their protein targets. A combination of affinity or activity data and energetically accessible conformational ensembles, each conformer described by three-dimensional (3-D) sensitive descriptors, forms the basis of the TACS data model. Recursive pruning is used to reduce the size of both the conformational ensemble and the descriptor space until the TACS data model contains just enough information to determine probable conformation(s) of ligands bound to their protein targets. The TACS algorithm is comprised of five components: (1) conformational ensemble generation, (2) 3-D sensitive descriptor calculation, (3) ensemble descriptor preprocessing, (4) model generation, and (5) prediction of bound conformation(s). Significantly, this method precludes the need for subjective or objective molecular alignment. We report the application of this technique to five benchmark protein-ligand couples where the conformation of a bound ligand has been previously established using X-ray crystallography: 9-cis-retinoic (1) and 9-trans-retinoic acid (2), both agonists for the retinoic acid receptor gamma, compounds KH1060 (3) and MC1288 (4), which bind to the vitamin D3 receptor, and R04 (5), an inhibitor bound to human rhinovirus 14 thermolysin. The binding conformations predicted by TACS were compared to the crystallographic structures extracted from their respective binding sites using root-mean-squared deviation (rmsd) criteria. Three of the conformations found using TACS were within crystallographic error. 9-cis-Retinoic acid, 9-trans-retinoic acid, and MC1288, when superimposed on their crystallographic structures, gave rmsd values of 0.22, 0.17, and 0.34 A, respectively. The rmsd values for KH1060 (1.54 A) and R04 (1.01 A) were larger but still reasonable.

Binding Sites↗
PubMed2002

Activation of the Aspergillus PacC zinc finger transcription factor requires two proteolytic steps.

The Aspergillus PacC transcription factor undergoes proteolytic activation in response to alkaline ambient pH. In acidic environments, the 674 residue translation product adopts a 'closed' conformation, protected from activation through intramolecular interactions involving the < or = 150 residue C-terminal domain. pH signalling converts PacC to an accessible conformation enabling processing cleavage within residues 252--254. We demonstrate that activation of PacC requires two sequential proteolytic steps. First, the 'closed' translation product is converted to an accessible, committed intermediate by proteolytic elimination of the C-terminus. This ambient pH-regulated cleavage is required for the final, pH-independent processing reaction and is mediated by a distinct signalling protease (possibly PalB). The signalling protease cleaves PacC between residues 493 and 500, within a conserved 24 residue 'signalling protease box'. Precise deletion or Leu498Ser substitution prevents formation of the committed and processed forms, demonstrating that signalling cleavage is essential for final processing. In contrast, signalling cleavage is not required for processing of the Leu340Ser protein, which lacks interactions preventing processing. In its two-step mechanism, PacC processing can be compared with regulated intramembrane proteolysis.

Amino Acid Sequence↗