1 International Centre for Genetic Engineering and Biotechnology, Padriciano 99, Trieste 2 Department of General Chemistry, University of Pavia, Pavia, Italy, 3 Peking University, Beijing, China, 4 Hunan Normal University, Hunan, China and 5 Proteros Biostructures GmbH, Planegg-Martinsried, Germany
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Abstract |
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Keywords: amylase inhibitors/cis-prolines/disulfide bridges/knottins/protein structure
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Introduction |
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A major source of interest in disulfide-rich small proteins is their widespread distribution in Nature (e.g. plants, insects, mammals) as well as their extreme functional variability, ranging from enzymes to metal ion channel inhibitors. The knottin fold occurs both in monoglobular proteins (e.g. protease inhibitors or ion channel regulators) and in multidomain proteins where sometimes one finds several subsequent knot motifs within the same chain, such as in thrombomodulin, transforming growth factor or E-selectin (Harvey et al., 1991; Graves et al., 1994
; Meininger et al., 1995
).
Recently, the three-dimensional solution structure of a knottin protein, the -amylase inhibitor AAI, has been determined in solution by NMR spectroscopy (Lu et al., 1999
) along with its crystal structure in complex with an insect
-amylase (Barbosa Pereira et al., 1999
). This amylase inhibitor adopts a typical knottin fold (Figure 1
) with an abcabc disulfide topology, which means that the first cysteine in the protein sequence forms a disulfide bridge with the fourth, the second with the fifth and the third with the sixth. Several other enzyme inhibitors, mainly protease inhibitors, are known to assume such a compact structure: hirustasin (Mittl et al., 1997
; Uson et al., 1999
), antistasin (Lopatto et al., 1997
), carboxypeptidase inhibitor (Rees and Lipscomb, 1982
) and some trypsin inhibitors (Bode et al., 1989
; Chiche et al., 1989
; Huang et al., 1993
). The latter have been shown to resemble structurally several other proteins of different biological functions (Pallaghy et al., 1994
).
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Methods |
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Results and discussion |
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The backbone conformation of the -amylase inhibitor in solution and in complex with the Tenebrio molitor
-amylase is shown in Figure 1
. Upon binding to the amylase, the structure of the
-amylase inhibitor becomes more compact: (i) the solvent-accessible area decreases from 2648 to 2406 Å2, (ii) the volume of the inhibitor decreases from 2909 to 2806 Å3 and (iii) the number of hydrogen bonds, identified with the WHAT IF program (Vriend, 1990
), increases from 12 to 21. Nevertheless, the two structures superpose fairly well (r.m.s.d. between all equivalent C
= 0.78 Å).
The major difference between complexed and free -amylase inhibitor is the isomerization of Pro20, which is trans in the free and cis in the complexed form. As the cis peptide bond is more constrained than the trans (Weiss et al., 1998
; Jabs et al., 1999
; Pal and Chakrabarty, 1999), this feature is likely to confer a higher rigidity to the complexed form. As a consequence of the transcis isomerization, the polypeptide segment 1322 located between the first and the second ß-strands (110 and 2430, respectively) undergoes a major rearrangement. This is shown quantitatively by the fact that if only the C
of residues of 110 and 2430 are superposed (r.m.s.d. = 0.48 Å), the segment 1322 shows a remarkable deviation (r.m.s.d. = 1.24 Å) (Figure 1
). Furthermore, the two disulfide bridges close to Pro20 undergo substantial conformational rearrangements (Figure 2
and Table I
), although this could also reflect the lower accuracy of 1H NMR experiments in determining the SS stereochemistry (Fletcher et al., 1997
). Interestingly, transcis-proline isomerization upon enzyme binding has been observed in another knottin-like protein, the protease inhibitor hirustasin (Mittl et al., 1997
; Uson et al., 1999
).
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In a search for similar structures, we analyzed all proteins of the Protein Data Bank (Bernstein et al., 1977) that have, at least three disulfide bridges in an abcabc topology which is identical with the amaranth
-amylase inhibitor. In structures with more than three disulfide bonds, the topology of all possible subsets of three SS bridges was examined. The retrieved data were then inspected visually in order to eliminate redundancies. From the structures of identical chain(s) and identical crystal space groups, the one with the best crystallographic resolution was retained. Single point mutants were rejected, while both the crystallographic and the NMR structures of the same protein were considered, where appropriate, since it is not obvious that structures in different physicochemical phases are identical. This analysis yielded a data set of 84 structures of domains with abcabc topology.
The similarity between two structures within this data set was then estimated as the root-mean-square distance between the aligned cysteine atoms computed after their optimal superposition. An alternative distance measure based on the superposition of all the C atoms, was also computed. Since the two approaches resulted in analogous results, those given by the first strategy are reported and commented on here. A cluster analysis was performed on a proximity matrix in which each element x8ij is an r.m.s.d. value between the structures i and j calculated in the above-indicated manner. The dendrogram resulting from the cluster analysis is shown in Figure 5
. The r.m.s.d. value corresponding to the optimal number of partitions is about 1.61.8 Å since the plot of the number of clusters versus the threshold of similarity, under which two clusters merge, was found to show a clear edge around these values (data not shown) (Malinowski, 1991
)
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Members of cluster 5 have a variety of origins and biological roles, but all of them seem to be the ligands, most often inhibitors, of other proteins, such as voltage-sensitive ion channels and enzymes. They are found in venom of snails and spiders and in plant tissues. The most typical representatives of cluster 5 are various conotoxins, toxins present in the venom of piscivorous marine snails which interact selectively with the various types of voltage-sensitive calcium, sodium and potassium channels, either in neurons or in skeletal muscles (Catterall, 1988;Gray et al., 1988
; McCleskey et al., 1988
; Olivera et al., 1988
; Yanagawa et al., 1988
; Terlau et al., 1996
). They have pharmacological applications (Miljanich and Ramachandran, 1995
) and have been used to characterize membrane channel assemblies (Tsien et al., 1991
; Olivera et al., 1994
). Cluster 5 also contains several neurotoxins present in the venom of spiders. Huwentoxin was shown to interact with the nicotinic acetylcholine receptor (Zhou et al., 1997
).
-Atracotoxin-HV1 (Atkinson et al., 1993
),
-agatoxin IVA and
-agatoxin IVB interact with voltage-sensitive calcium-channels (Regan, 1991
; Mintz et al., 1992
). Voltage-sensitive sodium channels interact with
-atracotoxin-HV1 (Nicholson et al., 1994
), robustoxin (2Mylecharane et al., 1989
) and µ-agatoxin-I (Skinner et al., 1989
). Interestingly, some of these toxic polypeptides have been shown to possess remarkable specificity. For example,
-atracotoxin-HV1 acts on insects but not on mammalian neuronal calcium channels (Fletcher et al., 1997
).
-Atracotoxin-HV1 is severely toxic towards newborn mice and primates but not other vertebrates (Mylecharane et al., 1989
; Nicholson et al., 1994
).
A further member of cluster 5 is gurmarin, a protein extracted from leaves of Gymnema sylvestre, an indian plant whose leaves were chewed as a folkloric treatment for diabetes mellitus (Imoto et al., 1991). Gurmarin is known to suppress specifically the sweet taste sensation in rats (but not in humans) (Katsukawa et al., 1999
). It is thermostable and supports both high pH and urea concentrations, but its biological role is unknown at a molecular level. It has recently been reported that gurmarin has no effect on several voltage-sensitive ion channels (Fletcher et al., 1999
).
In the proteins of cluster 5, the residues important for partner recognition and activity have been convincingly identified only in very few cases. Residues important for activity have been identified by site-directed mutagenesis studies on -conotoxin GVIA (Kim et al.,1994; Nadasdi et al., 1995
; Lew et al., 1997
). Nevertheless, in most cases, the relative importance of various residues is merely speculative. The action mechanism of gurmarin is unknown. At least two hypotheses have been proposed for the action mechanism of
-conotoxin PVIIA (Savarin et al., 1997
; Scanlon et al., 1997
).
In an attempt to detect possible similarities in the three-dimensional arrangement of residues important for activity, all structures of this group were superposed on that of the complexed amaranth -amylase inhibitor, by considering the C
atoms. Care was taken to treat separately the main and side chains, since it is not necessarily true that the main and side chains of a given residue of the
-amylase inhibitor best superpose the main and side chains of the same residue in another structure. The results (see Table IV
) clearly indicate that there is little three-dimensional conservation among the members of this group. For example, some important residues of the
-amylase inhibitor never superpose on residues important for activity in other proteins of cluster 5. The mean three-dimensional position conservation is only 11 and 14% for the main and side chains, respectively.
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The members of cluster 5 fold with a triple-stranded ß-sheet stabilized by three disulfide bonds (Figure 1). The lengths of the chains and of the three ß strands and also the length of the polypeptide segments separating the six cysteines are variable (Tables V and VI
). The protein core is formed by only 620% of the residues. The mean fractional solvent accessibility is very high, ranging from 1.22 to 1.56. For comparison, in proteins of 100200 amino acids, the core comprises about 40% of the residues and the mean fractional solvent accessibility is usually around 0.70. It is therefore not surprising that these small proteins, despite the presence of three disulfide bridges, experience a high mobility. In
-conotoxin MVIIC, for example, there are only three slowly exchanging amide protons (Farr-Jones et al., 1995
). The flexibility is not restricted to the loops. In some cases, such as in
-conotoxin PVIIA (Savarin et al., 1997
) and conotoxin GS (Hill et al., 1997
), even the disulfide bridges have been observed in multiple conformations. We mention that that some of these findings could reflect the difficulty of defining disulfide bond geometries from 1H NMR data (Fletcher et al., 1997
).
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Since there is no reason to suppose that the structures of cluster 5 are more accurate than other protein three-dimensional structures, it must be concluded that the high frequency of cis-prolines together with the high proline content is an essential feature of these proteins. It has been shown that cis peptide bonds restrict the conformational space available (Jabs et al., 1999) and it therefore reasonable that structures of cluster 5 employ cis-prolines to stabilize their fold.
All the three features described above discriminate the members of cluster 5 from the other knottin domains. Post-translational modifications are less frequently observed within the other knottins, only the domains of clusters 3 and 7 have an unusual high frequency of prolines and only in cluster 3 these residues quite often adopt a cis backbone conformation.
Conclusions
Upon binding to -amylase, the AAI molecule adopts a compact conformation which is characterized by, among others, a trans to cis isomerization of Pro20. This isomerization is likely to imply subtle but relevant consequences: (i) the complexed inhibitor becomes conformationally more constrained; (ii) two of the three disulfide bridges are forced to adopt different conformations relative to the uncomplexed inhibitor; and (iii) the consequent displacement of some side chains is likely to favor the reorientation of other, adjacent side chains, which thus become optimally oriented towards the amylase sites that they must recognize.
A systematic analysis of the 3-D structure databank revealed several structural clusters among proteins and domains that share the abcabc disulfide topology of AAI. Interestingly, the proteins that cluster together with AAI have a variety of evolutionary origins, but the same as AAI they have a relatively high proline content and many of them contain cis-proline residues. We therefore conclude that the cis-Pro may be a structurally important feature of this group of proteins.
The structural comparison of the knottin proteins revealed large variations among the members of this group. Little conservation is seen in terms of surface electrostatics and among the functionally important residues. As a consequence, it appears that a common evolutionary origin cannot be suggested from these data. In other terms, the knottin fold in general may have emerged as the result of convergent evolution.
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Notes |
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References |
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Received May 9, 2000; revised February 26, 2001; accepted June 18, 2001.