Analysis of the sequence specificity of SecA for peptide binding 106

To catalyze translocation, SecA must interact with a variety of unfolded polypeptides, and the degree of sequence specificity for the interaction could therefore be relatively low. This was the rationale for using the complete sequence of a translocation substrate (MBP) for the initial SPR experiments. The use of oriented peptide libraries to find specific short sequences that bind to SecA facilitated the creation of a sensor chip that displayed a single peptide sequence from the FhuD protein, immobilized in a defined manner. The results using the immobilized FhuD peptide were fully consistent with the results from the immobilized MBP CNBr digest. This indicated that whatever was happening during the binding reaction with the MBP digest was also happening with a more well-defined system. The results from the oriented peptide libraries also indicate that SecA does exhibit a degree of sequence specificity. We have used the results of these experiments, which were carried out prior to my arrival in the laboratory, to analyze in detail the sequence specificity of the SecA-peptide interaction.

The oriented peptide libraries consisted of 15 mer peptides bound to a cellulose sheet by a flexible linker attached to their C-terminus. The sequences of the peptides corresponded to the sequences of exported preproteins as well as resident cytoplasmic proteins. The arrays were used to “scan” through protein sequences. For example, preMBP (396 residues) was represented by approximately one hundred 15-mer peptides that started at the N-terminal sequence and were shifted by 4 residues until the last peptide that constitutes the extreme C-terminal sequence. A fluorescently labeled SecA molecule was used to measure binding to the immobilized peptides. After removal of the background, each peptide spot was scored for SecA binding in terms of a “relative signal index” (RSI), which ranged from 0 for the weakest binding to 1 for the strongest binding.

To determine the optimal sequence for relatively strong binding to SecA, the peptides (approximately 1500) were ranked according to their RSI and divided into two groups. Group 1 represented peptides that had a RSI > 0.4 (78 members), and Group 2, that had a RSI > 0.5 (47 members). Lists of the peptides in the two groups are provided in the Appendix B.

Alignment of the peptides by common multiple sequence alignment tools did not result in a good alignment (data not shown). Instead, more specialized tools for alignment and clustering of peptide data were used, such as GibbsCluster-1.1. The top-scored clusters resulted by this method are shown in Figure 3-11. The results of the clustering these peptides are shown the two different formats (Shannon style and Kullback-leibler style) for better observation of the patterns. As it can be seen, in the group A, the Lys/Arg residues are common at the C-termini of the polypeptides and the position -3 relative to the Arg/Lys residue is a hydrophobic amino acid (L/V/I/A/F). Analysis of the group 2 (peptides with higher binding strength) revealed a better- resolved pattern of amino acids. In these peptides Arg/Lys is located at the C-terminal of the pattern, and the two hydrophobic residues (L/A/V/I) are located at position -3 and -6 of the Arg/Lys residue. Based on these data it seems that the common sequence between the SecA binder peptides is [V/I/A/L/F/M]xx[V/A/I/L/F/M]xx[R/K].

Furthermore, the alignment of the 25 top peptides that had shown the highest binding strength towards the SecA proteins in the oriented peptide library experiments and share a common pattern is shown in Figure 3-12. It can be seen that binding motif is less that 8 residues in length. The Lys/Arg residues are highly conserved at the C-terminal of this motif followed a relatively conserved hydrophobic residue at -3 position relative to the Arg/Lys residue.

The predicted “SecA binding” sequence ([V/I/A/L/F]xx[V/A/I/L/F/M]xx[R/K]), which was derived from the alignment of top SecA-binder peptides, is relatively simple and can be found in many proteins. For example, on the MBP sequence, this motif has been repeated at least nine times (Figure 3-13). Analysis of other preproteins (such as OmpA, DegP, and GBP) using specialized tools in finding “patterns” in protein sequences (e. g. Protein Pattern Find) showed a similar result. The abundance of this “motif” on the protein sequences is consistent with the fact that SecA has to interact with numerous substrates in vivo that do not share a high degree of sequence identity.

A B

C D

C D

Figure 3-11. Alignment of top SecA-binder peptides

The peptides that showed the highest SecA binding strength were aligned. Top binders were organized into two groups, the first with relative signal intensity (RSI) > 0.4 and the second with RSI > 0.5. The alignment of the RSI > 0.4 group is shown in Panels A and B. A and B are essentially the results of a single alignment test, which are presented in two different formats for better observation of consensus sequences (A is presented in Kullback-leibler style, while B is in Shannon style). Panels C and D show the results of alignment of peptides with RSI > 0.5, and are represented in two different formats, similar to A and B.

---FIRSMKPRFVKRGAR--- ---TVDEALKDAQTRITK--- ----LVVSTLNNPFFVSLK--- ---MKKLKLHGFNNLTKS--- ---LLAEITPDKAFQDKL--- ---FEQAMQTRVFQPLKL--- ---TAEHTQSVLKGFNKF--- ---TIEKQLARTQRDKKR--- ---KVQAKYPVDLKLVVK--- ----LKSDVLFNFNKATLK--- ---LISKGIPADKISARG--- ---EIPALDKELKAKGKS--- ---AQYEDFIRSMKPRFV--- ----LIEWLPGSTIWAGKR--- ----EAVNKDKPLGAVALK--- ----EVVSHIASDNVLGGK--- ---ANIPVITLDRQATKG--- ----ENGAYKAQGVQLTAK--- ----NIDVGFGKLSLAATR--- ----ETADKVLKGEKVQAK--- ---ELANVQDLTVRGTKI--- ----LNNPFFVSLKDGAQK--- ---NGSDNKIALAARPVK--- ---SRRERSLRRLEQRKN--- ----SALTTMMFSASALAK--- x*xx*xx*

Figure 3-12. Location of the identified motif in the top SecA binder peptides.

The top 25 peptides that had shown the strongest binding towards SecA and shared a common pattern. The position of the conserved K/R residues, -3 hydrophobic residues, and -6 hydrophobic residues are highlighted with blue, black, and gray respectively.

>MBP|P0AEX9|27-396 KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSG LLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFN LQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAM TINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKP LGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTRITK >OmpA|P0A910|22-346 APKDNTWYTGAKLGWSQYHDTGFINNNGPTHENQLGAGAFGGYQVNPYVGFEMGYDWLGRMPYKGSVENG AYKAQGVQLTAKLGYPITDDLDIYTRLGGMVWRADTKSNVYGKNHDTGVSPVFAGGVEYAITPEIATRLEYQWT NNIGDAHTIGTRPDNGMLSLGVSYRFGQGEAAPVVAPAPAPAPEVQTKHFTLKSDVLFNFNKATLKPEGQAAL DQLYSQLSNLDPKDGSVVVLGYTDRIGSDAYNQGLSERRAQSVVDYLISKGIPADKISARGMGESNPVTGNTCD NVKQRAALIDCLAPDRRVEIEVKGIKDVVTQPQA >GBP|P0AEE5|24-332 ADTRIGVTIYKYDDNFMSVVRKAIEQDAKAAPDVQLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAG TVIEKARGQNVPVVFFNKEPSRKALDSYDKAYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKG EPGHPDAEARTTYVIKELNDKGIKTEQLQLDTAMWDTAQAKDKMDAWLSGPNANKIEVVIANNDAMAMGAVEA LKAHNKSSIPVFGVDALPEALALVKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVP YVGVDKDNLAEFSKK

Figure 3-13. The SecA-binding motif position along the preprotein substrates

The sequences of the mature regions of several E. coli SecA preprotein substrates were retrieved from Uniprot, and using the Protein Pattern Find tool, the location the “SecA binding motif”, [V/I/A/L/F]xx[V/A/I/L/F/M]xx[R/K], was identified. The identified sites that match this motif are highlighted in yellow. The results for MBP, OmpA, and GBP are provided here.