Protein-Protein Recognition Control by Modulating Electrostatic Interactions Song Han,†,# Shijin Yin,†,# Hong Yi,†,# Ste´phanie Mouhat,‡ Su Qiu,† Zhijian Cao,† Jean-Marc Sabatier,‡ Yingliang Wu,*,† and Wenxin Li*,† State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei 430072, P. R. China, and ERT 62 ,Inge´nierie des peptides a` vise´e the´rapeutique., Universite´ de la Me´diterrane´e - Ambrilia Biopharma S.A., Faculte´ de Me´decine Nord, Boulevard Pierre Dramard, 13916 Marseille Ce´dex 20, France Received January 12, 2010
Protein-protein control recognition remains a huge challenge, and its development depends on understanding the chemical and biological mechanisms by which these interactions occur. Here we describe a protein-protein control recognition technique based on the dominant electrostatic interactions occurring between the proteins. We designed a potassium channel inhibitor, BmP05-T, that was 90.32% identical to wild-type BmP05. Negatively charged residues were translocated from the nonbinding interface to the binding interface of BmP05 inhibitor, such that BmP05-T now used BmP05 nonbinding interface as the binding interface. This switch demonstrated that nonbinding interfaces were able to control the orientation of protein binding interfaces in the process of protein-protein recognition. The novel function findings of BmP05-T peptide suggested that the control recognition technique described here had the potential for use in designing and utilizing functional proteins in many biological scenarios. Keywords: protein-protein control recognition • electrostatic interactions • potassium channel • molecular engineering
Introduction Protein-protein interactions are responsible for the regulation and complexity of biological systems by forming macromolecular complexes and networks. Theory and methods that interfere with protein-protein interactions are desirable, as they offer great promise for manipulating biological networks and thereby guiding biotechnological applications. On the basis of known protein-protein recognition ways, combined computational and experimental methods have been successfully used to design proteins for a specific purpose.1,2 However, the ability to rationally create novel protein-protein interactions remains a significant challenge. The primary difficulty in the control of protein-protein recognition is defined by our inability to understand specific mechanisms of molecular recognition, especially in terms of details related to the differential use of both binding and nonbinding interfaces of the protein. Widespread experimental and theoretical work has indicated that dominant electrostatic interactions, likely due to the polarity of protein surfaces, play important functional roles in mediating many protein-protein interactions, including antibody-antigen, enzyme-inhibitor, potassium channel-peptide inhibitor, and so forth.3-7 Progress has * To whom correspondence should be addressed. Tel: ++86-(0)-2768752831. Fax: ++86-(0)-27-68752146. E-mail:
[email protected] (Y.W.) and
[email protected] (W.L.). † Wuhan University. # These authors contributed equally to the work. ‡ Universite´ de la Me´diterrane´e - Ambrilia Biopharma S.A.
3118 Journal of Proteome Research 2010, 9, 3118–3125 Published on Web 04/21/2010
been made in terms of understanding the structure-function characteristics of the binding interfaces, and less attention has been given to investigating the functional roles of nonbinding interfaces. In this report, we focus on the functional roles of the nonbinding interfaces in electrostaticmediated protein-protein interactions. We found that the charged residues of the nonbinding interfaces controlled protein-protein recognition, which was illustrated by a change in the protein epitope after transposition of charged residues within the protein molecules. These findings are important for the design and application of novel functional proteins.
Materials and Methods The Model System of Protein-Protein Recognition. We chose the potassium channel-peptide inhibitor complex as our experimental system for several reasons: (i) In the binding interfaces, there are many negatively charged residues in the potassium channel vestibules and many positively charged residues in the peptide inhibitors from the scorpion venom. (ii) From experimental and theoretical work, dominant electrostatic interactions are found to mediate recognition of potassium channel-peptide inhibitor interactions.5,8-11 (iii) The structures of two classical potassium channels (KcsA and Kv1.2) were resolved.12,13 (iv) Many determined structures of peptide inhibitors from scorpion venom have excellent structural stability, whose R-helix and β-sheet motifs are cross-linked by three or four disulfide bridges (Figure 1).14 Importantly, these characteristics have been implemented in the past to 10.1021/pr100027k
2010 American Chemical Society
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Protein Control Recognition by Electrostatic Interaction
Figure 1. The model system of protein-protein recognition and design strategy. (A) Structural characteristics of potassium channel peptide inhibitors and design of the protein binding interface switch. The peptide R-helix and β-sheet motifs are cross-linked by the disulfide bridges (solid line). Positive residues are shaded in light blue, and negative residues are colored pink. Green asterisks indicate the design changes between BmP05 and BmP05-T. (B) Structural stability of the BmP05-T peptide. The circular dichroism spectra of ScyTx (PDB code: 1SCY), BmP05, and BmP05-T peptides. The measurement was carried out in the UV range of 250-190 nm at 25 °C in water on a Jasco-810 spectropolarimeter at a concentration of 0.65-0.7 mg/mL. (C) Differential binding interfaces between ADWX-1 and ScyTx inhibitors. (D) The hypothesis of the binding interface switch between BmP05 and designed BmP05-T peptides.
develop a template for designing novel CD4 mimics to inhibit HIV-1 entry.15 Peptides and Potassium Channels. ScyTx peptide was provided by Latoxan (http://www.latoxan.com/). BmP05, BmP05T, and BmP05-T-K20V/K25A peptides were synthesized according to a previously described procedure,16 and the other BmP05-T mutants were produced as described previously.8 The cDNAs encoding mKv1.1, mKv1.3, hSKCa2, and hSKCa3 were generously provided by Prof. Stephan Grissmer (University of Ulm, Ulm, Germany), Prof. Nipavan Chiam-Vimonvat (University of California, Davis, CA), and Prof. George Chandy (University of California, Irvine, CA). Analysis of Peptide Binding Properties by Electrophysiological Recordings. Currents from COS-7 cells expressing SKCa channels and HEK293 cells expressing voltage-gated K+ channels were measured by the whole-cell patch-clamp technique using an EPC 10 patch clamp amplifier (HEKA Elektronik, Lambrecht, Germany) controlled by PULSE software (HEKA Elektronik). Internal pipet and external solutions were prepared as previously reported.8 The SKCa currents were elicited by 1 µM internal calcium and 200 ms voltage ramps from -140 to +40 mV at the holding potential of 0 mV. The voltage-gated K+ channel currents were elicited by depolarizing voltage steps of 200 ms from the holding potential -80 mV to +50 mV. Inhibitory peptides were dissolved in stock solutions containing 1% BSA and diluted into bath solutions containing 0.01% BSA for the determination of peptide binding affinity in electrophysiological experiments. Data were reported as the mean ( SD of at least three experiments. Statistic analyses were performed with the Student’s t test. Differences in the mean values were considered significant at p < 0.05. Concentration-response relationships were fitted according to the modified Hill equation: Itoxin/Icontrol ) 1/1 + ([peptide]/IC50), where I is the peak current and
[peptide] is the concentration of peptides, using IGOR software (WaveMetrics, Lake Oswego, OR). The parameters to be fitted were concentration of half-maximal effect (IC50). Determination, Modeling, and Analysis of Protein Structures. The secondary structure of BmP05-T and its mutants were measured by Circular Dichroism (CD) spectroscopy. Measurements were carried out in the UV range of 250-190 nm at 25 °C in water on a Jasco-810 spectropolarimeter at a concentration of 0.2-0.4 mg/mL. For each peptide, spectra were collected from three separate recordings and averaged after subtracting the blank spectrum of pure water. The structures of the mKv1.3 channel, BmP05, and BmP05-T were modeled using the closed-state KcsA channel (PDB code: 1BL8) and ScyTx (PDB code: 1SCY) as templates through the SWISS-MODEL server (http://swissmodel.expasy.org/). Using the modeled structures, ZDOCK17 program was used to generate the candidate complex structures. Through clustering analysis with the mutagenesis results, some possible hits were screened out, followed by a 500-steps energy minimization and 500 ps unrestrained molecular dynamics performed on each candidate complex to examine their structural stabilities. The final peptide-channel complex was sufficiently equilibrated by molecular dynamic simulations to introduce enough flexibility for both the receptor and the ligand. The interactive energies for BmP05 and its mutants with the Kv1.3 channel were calculated by using AMBER 8 package.18,19 More details were described in the previous study.8,11
Results Design Strategy for Protein Control Recognition. In this work, we selected potassium channel-peptide inhibitor complex as our system. We chose not to modify the potassium channel protein in order to alter the interaction between it and Journal of Proteome Research • Vol. 9, No. 6, 2010 3119
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Figure 2. Novel binding properties of the designed BmP05-T peptide. (A-D) Current traces in the absence (control) or presence of wild-type BmP05 peptide on SKCa2, SKCa3, Kv1.1, and Kv1.3 channels. (E-H) Current traces in the absence (control) or presence of designed BmP05-T peptide on SKCa2, SKCa3, Kv1.1, and Kv1.3 channels. (I) Concentration-dependent binding of the designed BmP05-T peptide to SKCa2 and SKCa3 channels. (J) Concentration-dependent binding of the designed BmP05-T peptide to Kv1.1 and Kv1.3 channels. Data represent the mean ( SD of at least three experiments.
its inhibitor, as it is located in the cell membrane. Instead, we designed an inhibitory peptide that uses the nonbinding interface of the wild-type peptide as the binding interface for interaction with the potassium channel. As shown in Figure 1C, peptide inhibitors ScyTx and ADWX-1 primarily use R-helix and β-sheet domains as binding interfaces, respectively.8,10 Likewise, we observed that the nonbinding interfaces of the ScyTx and ADWX-1 peptides contained several negatively charged residues, whereas the binding interfaces contained a large number of positively charged residues, resulting in a highly polar molecule (Figure 1A,C). Therefore, we hypothesized that the negatively charged residues oriented the inhibitor peptides to properly recognize the potassium channel protein. On the basis of the hypothetic correlation between binding interfaces and the characteristic distribution of charged residues within peptide inhibitors, we used the BmP05 peptide as our template, as it primarily uses the R-helix domain as the binding interface (Figure 1D).20 We designed a novel BmP05-T peptide by simultaneously replacing Gln9, Asp24, and Glu27 of the BmP05 peptide with Glu9, Val24, and Lys27 in BmP05T, respectively. As shown in Figure 1A, BmP05-T is 90.32% identical to BmP05, and importantly retained three essential residues (Lys6, Arg7, and Arg13) that are present on the binding interface of BmP05.20 Also, the acidic residue transposition between the BmP05 β-sheet domain and the BmP05-T R-helix 3120
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domain did not affect the structural stability of the peptide, as demonstrated by the CD spectra among homologous ScyTx (PDB code: 1SCY), BmP05, and BmP05-T peptides (Figure 1B). After transposition of negatively charged residues between the BmP05-T and BmP05 peptides, the molecular polarity of BmP05-T was changed to resemble that of the ADWX-1 inhibitor (Figure 1D), where it used the β-sheet domain as the binding interface. BmP05 peptide mainly used R-helix domain as the binding interface (Figure 1D), and would designed BmP05-T use β-sheet domain instead of R-helix domain as the binding interface? If so, the functional residues in the BmP05 binding interface would become less effective on binding to potassium channels in the designed BmP05-T peptide; on the contrary, the nonfunctional residues in the BmP05 nonbinding interface would become significant effective on binding to potassium channels in the designed BmP05-T peptide. The switch event between the binding and nonbinding interfaces was further verified in the following experiments. The Binding Properties of BmP05-T. As illustrated in Figure 1, the BmP05-T peptide showed high sequence and structural similarity to the BmP05 peptide, but had different molecular polarity. To test if transposition of negatively charged residues in BmP05-T resulted in different binding properties of the peptide as compared to wild-type BmP05, we first identified the activity profiles of the BmP05 peptide on different potassium channel subtypes. Similar to the homologous ScyTx
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Table 1. Effect of BmP05-T and Its Mutants in Blocking Kv1.3 Channelsa BmP05-T mutant
IC50 (nM)
n
IC50 (mut)/ IC50 (wt)
BmP05-T BmP05-T-K6A BmP05-T-R13A Bmp05-T-k20A BmP05-T-K25A BmP05-T-K27A BmP05-T-K30A BmP05-T-K20 V/K25A
22.01 ( 3.9 246.26 ( 12 47.42 ( 8.59 221.96 ( 38.1 1200.4 ( 263 448.17 ( 80.9 308.14 ( 45.2 8580 ( 3230
6 6 5 6 6 5 5 6
1.0 11.2 2.2 10.1 54.5 20.4 14.0 389.8
a Each value represents mean ( SD. n, number of individual experiments.
peptide (Figure 1A),21 BmP05 was also specific for small conductance calcium-activated K+ (SKCa) channels, where 0.1 nM and 1 nM of BmP05 peptide inhibited SKCa2 and SKCa3 current by about 50%, respectively (Figure 2A,B). The activity of Kv1.1 and Kv1.3 was not significantly altered to significant levels in the presence of 10 µM BmP05 (Figure 2C,D). However, the designed BmP05-T peptide possessed binding properties distinct from those of the BmP05 peptide. As shown in Figure 2E-H, BmP05-T was no longer specific for the SKCa channel. For example, BmP05-T had significantly lower binding affinities for SKCa2 and SKCa3 channels, as indicated by moderate blocking of potassium current with 100 nM of BmP05-T. On the basis of the concentration-response curve for SKCa channels in Figure 2I, the IC50 values of BmP05 binding were 69.07 nM and 130.6 nM for SKCa2 and SKCa3, respectively. Likewise, BmP05-T bound more tightly to Kv1.1 and Kv1.3 (Figure 2G,H), where 300 and 30 nM effectively blocked their potassium current, respectively, and the concentration-response curve indicated that the IC50 values of BmP05-T binding were 160.07 and 22.01 nM, respectively (Figure 2J). The fact that BmP05 and BmP05-T share similar structures yet possess different binding properties suggests that altered molecule polarity of the two peptides likely results in binding of potassium channels through distinct interfaces. Switch between the Binding and Nonbinding Interfaces. The sequence alignment between BmP05-T and BmP05 indicated that the residues Lys6, Arg7, and Arg13 were retained in the R-helix domain of BmP05-T (Figure 1A).20 To test if BmP05-T no longer used the R-helix domain as the binding interface, we constructed two mutants, BmP05-T-K6A and BmP05-T-R13A. In addition, we selected the Kv1.3 channel to determine the binding properties of the BmP05-T mutants because it was the most sensitive to BmP05-T binding among the four channel subtypes (Figure 2). In comparison with the binding affinity of BmP05-T toward the Kv1.3 channel (IC50 ) 22.01 nM), BmP05-T-K6A and BmP05-T-R13A had comparable binding activity to that of BmP05-T, where the IC50 values for BmP05-T-K6A and BmP05-T-R13A were 246.26 nM and 47.42 nM, respectively (Table 1 and Figure 3A-C). Meanwhile, there were few structural differences between these mutant peptides, as evidenced by the similar CD spectra among BmP05-T, BmP05-T-K6A, and BmP05-T-R13A (Figure 3D). The structural and functional similarities between BmP05-T and these mutants strongly indicate that BmP05-T could use a binding interface distinct from that of the BmP05 peptide. To investigate if BmP05-T uses β-sheet domains as the binding interface, we next constructed BmP05-T-K20A and BmP05-T-K25A mutants. BmP05-T-K20A had comparable bind-
Figure 3. Functional silencing of Lys6 and Arg13 residues in the R-helix domain of the designed BmP05-T peptide. (A and B) Current traces in the absence (control) or presence of BmP05T-K6A and BmP05-T-R13A mutants on the Kv1.3 channel. (C) Concentration-dependent binding of BmP05-T-K6A and BmP05T-R13A mutants to the Kv1.3 channel. The IC50 values are listed in Table 1. (D) The circular dichroism spectra of BmP05-T, BmP05T-K6A, and BmP05-T-R13A.
ing affinity to Kv1.3 and Kv1.1 as that of BmP05-T (Figure 4A,B). Binding of BmP05-T-K25A was much weaker, where the IC50 values of BmP05-T-K20A and BmP05-T-K25A binding were 221.96 and 1200.4 nM, respectively (Table 1 and Figure 4F). We also observed similar CD spectra among BmP05-T, BmP05T-K20A, and BmP05-T-K25A peptides (Figure 4E), suggesting that residue substitution did not affect structural stability of these mutants. Together, these data indicate that Lys25 is an important functional residue in the interaction between BmP05-T and potassium channels. Next, we constructed the double mutant BmP05-T-K20V/K25A. As shown in Figure 4C, the binding affinity of BmP05-T-K20V/K25A toward Kv1.3 was significantly decreased, where 10 µM of BmP05-T-K20V/K25A inhibited only about 50% of the potassium current. The IC50 value obtained for BmP05-T-K20V/K25A was 8580 nM, resulting in it being about 390-fold less sensitive than the wild-type BmP05-T peptide (IC50 ) 22.01 nM), further demonstrating that BmP05-T uses β-sheet domains as the binding interface. This finding was also supported by the change of binding affinity toward the Kv1.1 channel between BmP05-T and BmP05-TK20V/K25A, where the IC50 value obtained for blocking of the Kv1.1 channel by BmP05-T-K20V/K25A was 26.66 µM (Figure 4D), resulting in it being about 166-fold less sensitive than wildtype BmP05-T (IC50 ) 160.07 nM). Journal of Proteome Research • Vol. 9, No. 6, 2010 3121
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Figure 4. Functional importance of positively charged residues in the β-sheet domains of BmP05-T. Measurement was carried out in the UV range of 250-190 nm at 25 °C in water on a Jasco-810 spectropolarimeter with a concentration of 0.2-0.4 mg/mL.
We next studied the role of the two remaining lysine residues in the β-sheet domain by constructing the mutants BmP05-TK27A and BmP05-T-K30A. As illustrated in Table 1 and Figure 4G-I, these mutants showed decreased binding activities to Kv1.3 as compared to wild-type BmP05-T, where they were reduced by 20- and 14-fold for Lys27 and Lys30, respectively. In addition, the structures of both BmP05-T-K27A and BmP05T-K30A were significantly more stable, as judged by their CD spectra (Figure 4E). These data indicate that Lys27 is more important for BmP05-T binding to the Kv1.3 channel. Together, these results suggest that BmP05-T primarily uses β-sheet domains as the binding interface with the potassium channel instead of R-helix domains. In other words, the change in molecular polarity of the peptide induced a switch in the binding interface in BmP05-T through transposition of negatively charged residues. Structural Analysis of the Interaction between BmP05-T and Potassium Channel. Although BmP05-T is 90.32% identical to BmP05, it uses the opposite surface as compared to the BmP05 binding interface to recognize the potassium channel. We next investigated this switch in peptide binding interface by structural analysis. As shown in Figure 5A, there are five negatively charged residues in each subunit of Kv1.3, and 20 negatively charged residues on its binding interface. These residues form a strong negative electric surface on the outer entrance of the potassium channel (Figure 5B). As such, the negatively charged Glu9 residue in the BmP05-T peptide would induce a strong repulsion between BmP05-T and the Kv1.3 3122
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channel, inducing a change in orientation of BmP05-T as it approached the outer entrance of the Kv1.3 channel (Figure 5C). Reorientation of the BmP05-T molecule resulted in Glu9 being repositioned far away from the vestibule of the Kv1.3 channel (Figure 5D). Using computational techniques to illustrate protein-protein interactions,8 we next obtained a BmP05-T-Kv1.3 complex model. Three positively charged residues (Lys6, Arg7, and Arg13) of BmP05-T remained distant from the outer entrance of the Kv1.3 channel (Figure 5E), similar to negatively charged Glu9, and the replacement of two Lys6 and Arg13 by alanine had less effective on BmP05-T binding activities (Table 1 and Figure 3). Meanwhile, four positively charged residues (Lys20, Lys25, Lys27, and Lys30) of the BmP05-T peptide are positioned toward the outer entrance of the Kv1.3 channel, and the side chain of Lys25 is predicted to plug into the channel selectivity filter (Figure 5F). The recognition mechanism of these four positively charged residues is in agreement with the mutagenesis data (Table 1 and Figure 4), especially since Lys25 was found to be the most essential for BmP05-T binding of potassium channels. Importantly, the interactive energies for BmP05-T and its mutants interacting with Kv1.3 were calculated with previous methods.8 As shown in Table 2, the change of calculated binding free energies (∆∆Gbinding) agreed well with the experimental data of BmP05-T and its mutants (Table 1), which further highlight the importance of these positively charged residues in the switched binding interface with Kv1.3 channel (Figure 5). For example, the K25A and K20A/K25V mutants had much bigger ∆∆Gbinding
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Figure 5. Structural analysis of BmP05-T-Kv1.3 channel recognition. (A) Distribution of negatively charged residues on the binding interface of the Kv1.3 channel. Only two subunits of the Kv1.3 channel are shown for clarity. (B) Molecular surface rendering of the Kv1.3 binding interface. Negatively charged residues are colored red. (C and D) Orientation of BmP05-T with respect to Kv1.3. (E and F) Interaction details of charged residues of designed BmP05-T peptide. Table 2. Interactive Energies for BmP05-T and Its Mutants When Interacting with Kv1.3 Channela interactive energies
BmP05-T
K6A
R13A
K20A
K25A
K27A
K30A
K20A/K25V
∆Eelec ∆EvdW ∆∆Gbinding
-1742.70 -58.30 0
-1539.39 -58.00 0.27
-1525.66 -57.97 0.30
-1477.83 -57.88 0.08
-1293.24 -52.64 9.38
-1348.92 -55.11 4.39
-1496.47 -57.43 0.41
-1081.28 -54.05 11.53
a
All energies are in kcal/mol.
values of 9.38 and 11.53 kcal/mol, respectively (Table 2), which was in line with the fact that this mutation resulted in the much remarkable change in the affinity of BmP05-T to Kv1.3. Relatively, the replacement of Lys6 and Arg13 in the nonbinding interface slightly affected the BmP05-T activity, which agreed
well with the small ∆∆Gbinding values for these mutants (Table 2). When BmP05-T was bound to Kv1.3, the electrostatic interaction energies (∆Eelec) were far bigger than van der Waals interaction energies (∆EvdW) (Table 2), including BmP05-T and its mutants, showing that the electrostatic interaction energy Journal of Proteome Research • Vol. 9, No. 6, 2010 3123
research articles contributed dominantly to the recognition of BmP05-T peptide toward Kv1.3 channel. Among different BmP05-T mutants, the electrostatic effect of basic residues in BmP05-T binding interface was more significant for BmP05-T recognition than that of basic residues in BmP05-T nonbinding interface since these positively charged residues (Lys20, Lys25, Lys27, and Lys30) were adjacent to Kv1.3 channel (Table 2 and Figure 5). Together, the dominant effect of electrostatic interaction energies further rationalizes the switch of the BmP05-T binding interface compared with that of BmP05. In summary, the strong electric force between the designed BmP05-T peptide and the Kv3.1 potassium channel, and the molecular polarity of BmP05-T, dictates that the β-sheet domains of BmP05-T was used as the binding interface instead of the R-helix domain.
Discussion The control of protein-protein recognition remains a significant challenge in biotechnological applications. However, this field could be advanced if the molecular characteristics of both the binding and nonbinding interfaces of proteins were completely understood. There are typically three primary ways to mediate the protein-protein recognition process, including electrostatic interactions, van der Waals interactions, and synergetic effects between the two. In this work, we focused on the functional role of electrostatic-mediated protein-protein recognition at nonbinding interfaces, and were able to control the binding of a potassium channel to the designed BmP05-T inhibitor by changing residues within BmP05 that induced a switch in its molecular polarity (Figure 1). The designed BmP05-T peptide was 90.32% identical to the wild-type BmP05 peptide, and included the critical residues Lys6, Arg7, and Arg13, which are important for the activity of the BmP05 peptide. The most significant difference between BmP05-T and BmP05 is the location of specific negatively charged residues, including Glu9 in the R-helix domain of the BmP05-T peptide, and Asp24 and Glu26 in the β-sheet domains of the BmP05 peptide (Figure 1A). Subsequent structural and functional experiments demonstrated that Glu9 rotated onto the nonbinding interface of BmP05-T as it approached the binding pocket of the potassium channel. In other words, BmP05 primarily used the R-helix domain as the binding interface, whereas BmP05-T used the β-sheet domain (Table 1 and Figures 2-5). The changes in the binding interface observed here suggest a novel functional role for negatively charged residues in the nonbinding interfaces of these potassium channel inhibitors, as they were able to control the orientation of protein binding interfaces in the process of protein-protein recognition. The unique functional characteristics of BmP05-T (Figure 2) also showed that we were able to apply protein-protein control recognition for designing different functional proteins by adjusting the position of charged residues in the proteins themselves. To utilize the protein-protein control recognition technique described here in other biological scenarios, some important concerns should be noted. First, electrostatic interactions should be the dominant force that mediated the protein-protein recognition. Likewise, the designed protein was structural stable, as indicated by the structural similarity between BmP05-T and BmP05. Finally, there is a wide distribution of one kind of charged residues and a characteristic distribution of the other kind of charged residues. As shown in Figure 1, many positively charged residues widely distribute from the molecular N3124
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Han et al. terminal to the C-terminal end, and a few of negatively charged residues rarely distributed in the channel inhibitors. In conclusion, the technique of protein-protein control recognition described here has the potential for being widely applied in the field of protein chemistry, as there are many kinds of potassium channels and their various peptide inhibitors,22 and because they have been implicated in human disease.23 Likewise, this method could definitely accelerate the development of diagnostic and therapeutic agents specific for potassium channels and other proteins.8,23,24
Acknowledgment. This work was supported by grants from the National Natural Sciences Foundation of China (number 30530140, 30770519 and 30973636), and the National Basic Research Program of China (2010CB529800). References (1) Havranek, J. J.; Harbury, P. B. Automated design of specificity in molecular recognition. Nat. Struct. Biol. 2003, 10, 45–52. (2) Kortemme, T.; Joachimiak, L. A.; Bullock, A. N.; Schuler, A. D.; Stoddard, B. L.; Baker, D. Computational redesign of proteinprotein interaction specificity. Nat. Struct. Mol. Biol. 2004, 11, 371– 379. (3) Sheinerman, F. B.; Norel, R.; Honig, B. Electrostatic aspects of protein-protein interactions. Curr. Opin. Struct. Biol. 2000, 10, 153– 159. (4) Lee, L. P.; Tidor, B. Barstar is electrostatically optimized for tight binding to barnase. Nat. Struct. Biol. 2001, 8, 73–76. (5) Huang, X.; Dong, F.; Zhou, H. X. Electrostatic recognition and induced fit in the kappa-PVIIA toxin binding to Shaker potassium channel. J. Am. Chem. Soc. 2005, 127, 6836–6849. (6) Vizcarra, C. L.; Mayo, S. L. Electrostatics in computational protein design. Curr. Opin. Chem. Biol. 2005, 9, 622–626. (7) Kundrotas, P. J.; Alexov, E. Electrostatic properties of proteinprotein complexes. Biophys. J. 2006, 91, 1724–1736. (8) Han, S.; Yi, H.; Yin, S. J.; Chen, Z. Y.; Liu, H.; Cao, Z. J.; Wu, Y. L.; Li, W. X. Structural basis of a potent peptide inhibitor designed for Kv1.3 channel, a therapeutic target of autoimmune disease. J. Biol. Chem. 2008, 283, 19058–19065. (9) Thompson, J.; Begenisich, T. Electrostatic interaction between charybdotoxin and a tetrameric mutant of Shaker K+ channels. Biophys. J. 2000, 78, 2382–2391. (10) Wu, Y.; Cao, Z.; Yi, H.; Jiang, D.; Mao, X.; Liu, H.; Li, W. Simulation of the interaction between ScyTx and small conductance calciumactivated potassium channel by docking and MM-PBSA. Biophys. J. 2004, 87, 105–112. (11) Yi, H.; Qiu, S.; Cao, Z.; Wu, Y.; Li, W. Molecular basis of inhibitory peptide maurotoxin recognizing Kv1.2 channel explored by ZDOCK and molecular dynamic simulations. Proteins 2008, 70, 844–854. (12) Doyle, D. A.; Morais Cabral, J.; Pfuetzner, R. A.; Kuo, A.; Gulbis, J. M.; Cohen, S. L.; Chait, B. T.; MacKinnon, R. The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science 1998, 280, 69–77. (13) Long, S. B.; Campbell, E. B.; Mackinnon, R. Crystal structure of a mammalian voltage-dependent Shaker family K+ channel. Science 2005, 309, 897–903. (14) Rodriguez de la Vega, R. C.; Possani, L. D. Current views on scorpion toxins specific for K+-channels. Toxicon 2004, 43, 865– 875. (15) Martin, L.; Stricher, F.; Misse, D.; Sironi, F.; Pugniere, M.; Barthe, P.; Prado-Gotor, R.; Freulon, I.; Magne, X.; Roumestand, C.; Menez, A.; Lusso, P.; Veas, F.; Vita, C. Rational design of a CD4 mimic that inhibits HIV-1 entry and exposes cryptic neutralization epitopes. Nat. Biotechnol. 2003, 21, 71–76. (16) Mouhat, S.; Teodorescu, G.; Homerick, D.; Visan, V.; Wulff, H.; Wu, Y.; Grissmer, S.; Darbon, H.; De Waard, M.; Sabatier, J. M. Pharmacological profiling of Orthochirus scrobiculosus toxin 1 analogs with a trimmed N-terminal domain. Mol. Pharmacol. 2006, 69, 354–362. (17) Chen, R.; Li, L.; Weng, Z. ZDOCK: an initial-stage protein-docking algorithm. Proteins 2003, 52, 80–87. (18) Huo, S.; Massova, I.; Kollman, P. A. Computational alanine scanning of the 1:1 human growth hormone-receptor complex. J. Comput. Chem. 2002, 23, 15–27. (19) Case, D. A. Cheatham, T. A. D. T. E., III Simmerling, C. L. Wang, J. Duke, R. E. Luo, R. Merz, K. M. Wang, B. Pearlman, D. A. Crowley,
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