MpcT (Htr14) by itself probably serves as the ∆Ψ sensor

MpcT (Htr14) by itself probably serves as the ∆Ψ sensor

A high sequence homology between MpcT and its putative ortholog from N. pharaonis is present in the N-terminal but not in the C-terminal region

The question remains whether MpcT detects ∆∆Ψ directly or in conjunction with another protein or proteins. A known example of ∆∆Ψ sensing is the voltage-gated K+ _{channel KvAP}

from Aeropyrum pernix for which a positively charged but otherwise very hydrophobic "sensor paddle" domain was suggested to move perpendicular to the plane of the membrane upon a

∆∆Ψ, thereby inducing a conformational change in the gating part of the channel (Jiang et al., 2003).

One might speculate that ∆∆Ψ sensing via MpcT occurs by a similar mechanism, either involving a membrane-embedded segment of MpcT containing charged residues or via an interaction of MpcT with another protein containing such a segment. For the predicted TM regions (maybe in combination with some charged, also membrane-embedded residues from the N-terminal region) both options are possible, whereas the Pro-rich C-terminal region of MpcT would seem to require interactions with another protein or proteins. These two regions in MpcT are quite dissimilar in their primary sequence to the corresponding regions of other Htrs. Together with the linker region (between the second TM region and the putative coiled-coil region) they are proposed to be situated close to the membrane, as depicted in a hypothetical working model of an MpcT structure on the basis of a proposed structure of E. coli Tsr (Fig. 3.16).

When the amino acid sequence of MpcT was compared with that of its putative ortholog from the halophilic archaeon Natronomonas pharaonis (Michaela Falb et al., unpublished), no significant sequence identity could be found for the C-terminal region downstream of position 552 of MpcT (Fig. 3.14). However, in both proteins this region contains a stretch of 33 residues with a proline content of 21% in MpcT and 18% in its putative ortholog, which is unusually high

for Htrs. This might hint to a conserved function of this region, despite the poor sequence identity. The N-terminal region (up to position 166 of MpcT) is reasonably well conserved, with 43% identity, including all the prolyl residues (Fig. 3.15). The 34% identity found for the intermediate region (positions 167 to 552 of MpcT) is within the range seen when the corresponding intermediate regions of any two halobacterial transducers are compared. In addition to the putative coiled coil of methylatable and signaling regions (predicted to comprise

10 20 30 40 50 60 70 80 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

MNITQAYKRS LWWSMDMVGA TGSVERKMLT AV G LQ LAAF G GMAFLTVFTA G TVQLIGVGG MLALSVVAFY NTYLIAEADF

ttt tttttttttt tttttttttt hhhhtttttt tttttttttt tttttt 90 100 110 120 130 140 150 160 ...5671234 5671...| ....|....| ..1x345671 2x456712x4 56712x4567 1x3...| ....|....| V EPLVALEDA ADDIAAG EFE RADIPSSKRD DEIASLVASF DGMQSNLEVA SRQADALA RQ AFDD PAL DE S VPG AFG ESIT gdl-1 170 180 190 200 210 220 230 240 ...|12x45 67123X5671 2345671234 56712x4567 123456712| ....|....|.. ..|23456 7123456712

EMADS LEAYT AEL E DKTAEL EHQQAELERQ SEQLRALV D A LSEATDAARA GDL TATVDAA ALDVTDDHRA AVEDFNQLLE

gdl-2 250 260 270 280 290 300 310 320 3456712345 6712x45671 234X6712x4 5671xx4567 1234567123 4567123456 7123456712 3456712345 TLADTISDIQ SFSDAVLAVS RTTDERVDAV ADRSAAVSES VTEIA DGANQ QTNQLNNIA A EMDTVSA TVE E IAA S AND VA

mmm* *mmmm 330 340 350 360 370 380 390 400

6712x45671 234X671234 56712x4X67 12x4567123 45671234X6 71xx4X6712 345671x345 671x345671 KTAQAAADRG EDGRGEVEET IEALRALREQ SQAVAETVES LAAEVERIDG ITALIED I AE ETNMLALNAS I E AA RTGSDG ssss ssssssssss sssssshhhh 410 420 430 440 450 460 470 480 2x45671234 X671234X67 1x3456712x 4567123456 71x34X6712 3X56712x45 6712x45671 x345671234

DGFAVVADE V KDLA EE TREQ AADISEIV D A VTEKAEDASI AIGEVDAEVE RKITKAEGVL RDFEAIVDEV ANVNHAVQE I

ssssssssss sssss*s 490 500 510 520 530 540 550 560 5671234567 1234567123 4567123456 712345671x 3456712345 6712345671 xx45671234 56..|....| SDAT DQGAQS VTDVVGMV E E VASVSEETAA ESD TVA DNAA EQTDATDEVA DQMDE LA EQT AALA G MLDDF TVPADAGTA D

mmmm*mmm - pppppppp 570 580 590 600 610 620

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|.. QSVADDSPTA QPPAADDEPA AAVVDQPQPA SDAEDEEGVP DSGGESVAVS DGGWADDRSS FTWADSQ 627

pppppppppp pppppppppp pppppppppp pppppppppp

Figure 3.14 MpcT (Htr14) sequence with relevant features indicated.

MpcT contains an N-terminal stretch of 49 amino acids devoid of any charged residues. This sequence is proposed to constitute two transmembrane regions (t) connected by a hairpin loop (h). Regions that potentially contain a hydrophobic heptad motif, indicative of an amphipathic helix or a coiled-coil structure, are marked by attributing a succession of numbers 1 to 7 to the respective residues. Positions 1, 4 and 5 are highlighted green to indicate a good match if the corresponding residues are hydrophobic or are Ser or Thr, and they contain a green "X" if a charged residue is present. Positions 2 and 3 are highlighted magenta to indicate a good match if the corresponding residues are charged, and they contain a magenta "x" if a hydrophobic residue is present. Also indicated is the region of highest homology among all 18 Htrs, which represents the putative signaling region (s) with a hairpin turn (h) in its center. A C-terminal region is marked (p), which is unusually rich in prolyl residues (8/48), compared to the other Htrs. The positions of the two "GDL"-motifs, indicative of HAMP domains, are shown (gdl). Residues which match (m) or do not match (-) the E. coli transducer methylation consensus sequence are indicated at the major methylation sites. Big and small asterisks mark the glutamates which were identified via MS to be methylated to a high or low extent, respectively, within the MpcT population. An alignment of MpcT with its putative ortholog from

N. pharaonis identified residues which are identical in both proteins (bold underlining) or were substituted conservatively (weak underlining) according to the BLOSUM62 matrix.

residues 245 to 552 of MpcT), this intermediate region consists of an additional stretch of residues. This stretch comprises putatively α-helical regions (as deduced from the presence of hydrophobic heptad sequences), interrupted by a region of unknown structure, that contains a sequence which was named GDL-motif (Fig. 3.14). A GDL-motif was found to be indicative of the beginning of the "connector region" within a "HAMP domain" of a transducer. HAMP domains comprise a common structural element that was identified by sequence comparisons of sensor kinases and MCPs (Aravind and Ponting, 1999), and are frequently found in histidine protein kinases, adenylyl cyclases, methyl-binding proteins and phosphatases.

The N-terminal region of MpcT also exhibits significant homology to that of the putative H. salinarum histidine kinase OE2712R

A comparison of MpcT with proteins from H. salinarum strain R1 (Dieter Oesterhelt et al., unpublished; http://www.halolex.mpg.de) discovered protein OE2712R (the equivalent of protein Vng1193c from Halobacterium sp. NRC-1 (Ng et al., 2000)). OE2712R is 39% identical (from position 25 to 180) to the mentioned N-terminal region of MpcT (Fig. 3.15).

The C-terminal region of OE2712R (from position 183 to 392), however, is 32% identical to the putative histidine protein kinase (HPK) domain of halobacterial KinA (OE2961F, Vng1374g). His203 of OE2712R corresponds to the conserved phosphorylatable His405 of B. subtilis histidine kinase A (Wang et al., 2001). Assuming that the same signal is sensed by MpcT and OE2712R, these findings suggest that the N-terminal rather than the C-terminal regions of MpcT and OE2712R are responsible for the input of the ∆∆Ψ signal. In the case of MpcT, the output is likely to be via an interaction of its cytoplasmic signaling region with the associated HPK, CheA, which then relays the signal via the response regulator CheY to the flagellar motor as the final target. In the case of the putative sensor kinase OE2712R, the output might be a change in an intrinsic HPK activity and involve transcriptional regulation via an unknown response regulator. OE2712R is expected to form dimers just like Htrs or like other known HPKs, e.g. CheA. None of the other Htrs show a comparable similarity between a transducer region and a region from a putative sensor kinase.

Its genetic neighborhood points to an independent transcription of mpcT

Htr1, Htr2, CosT and BasT are the Htrs for which interactions with a photoreceptor or binding protein have been reported. All corresponding gene pairs are cotranscribed, and they either overlap or, in the case of htr2, are separated by just one base (Table 6.2). An inspection of the flanking sequences of mpcT revealed an AT-rich region, centered ca. 30 base pairs upstream of the coding region, that could represent a promoter sequence. This finding, together with the relatively large distance of 81 base pairs between mpcT and its neighboring upstream gene OE1537B1R (http://www.halolex.mpg.de), strongly argues that mpcT is not cotranscribed with an upstream gene. Furthermore, OE1537B1R encodes a small protein of 93 amino acids, which lacks TM regions and would therefore not meet the expected criteria for a ∆Ψ sensor. The nearest downstream gene, OE1534F (VNG0354C), is located 13 bp downstream, is in the opposite orientation and encodes a protein of 256 amino acids and 7 predicted TM regions. A A

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| Htr14 Hs ---MNITQAYKRSLWWSMDMVGATGSVERKMLTAVGLQFLAAGGMAFLTVFTAGTVQLIGVGGMLALSVV 67 Htr14 Np --MSSATGVRRWAGDIAAAYKRLLWGMMRVLNIDDSMWRKIMVAVGLQFGAGVALVATMLLLDGTVATAVGLTVFVLAAI 78 OE2712R MSHTQPTTTSDVLHRFWRRYESFIWWTMDAVGVASSVRAKILLAVTIQFAVSLAQVVVPVYFSGPARITAVGVLVVGATL 80 [ ttttttttttttttttttttttthhhhttttttttttttt ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| Htr14 Hs AFYNTYLIAEADFVEPLVALEDAADDIAAGEFERADIPSSK-RDDEIASLVASFDGMQSNLEVASRQADALARQAFDDPA 146 Htr14 Np AFFNTALIARDDLLQPLDEIRLSAEEISAGSLT-TDPPHIE-QDDEVGSLATSFERMHDTLQTTAKQADALANEEFDDPV 156 OE2712R ALVNTVLVVERDIVDPVTGLQAAATHIAEGDLAAGDLPRADGQQDEIGALVDDFAAMHSHLRVVAAQAEALADHDFDAAV 160 ttttttttt gdl-1 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| Htr14 Hs LDESVPGAFGESITEMADSLEAYTAELEDKTAELEHQQAELERQSEQLRALVDALSEATDAARAGDLTATVDAAALDVTD 226 Htr14 Np LDEEVPGAFGDSLDRMAENLSVAIADLEARSARLTELVDEFE---AACDRASDGDLTVRLSDEEVDDRF 222 OE2712R LETSLPGRFGASLRRMTASLDRYIDRITADRERSTLLNYLVSHDVPNVVNVLSGRLQLARRQTDAQAVRDHLDVADRQVS 240 ] * gdl-2

B

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| OE2712R GASLRRMTASLDRYIDRITADRERSTLLNYLVSHDVPNVVNVLSGRLQLARRQTD---AQAVRDHLDVADRQVSKIEEVC 246 KinA1 FSIVVDVTERAD-YEARLETQRDNLDVLNQVLRHDIRNDLQLVLAYADMLAGELDGEHADYIERVLESADHAVELTKTAR 447 * ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| OE2712R DVVGKLTRD-DGAAAIDVVTLLDSEVARVTESYPDAQVSADLPSPPCRVRGNELLNSVFRNLITNAVEHNDAPTPRVAVS 325 KinA1 DIADVMLTDPDADHVMSLRATLEAELAEVRSSFPSAAVTVDGSIPDVTVHGNNMLDSVFRNLLKNAIQHNDQDSPDVTVS 527 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| OE2712R VAVEAES-VTVRVADNGPGLDVSDSESFFQHR---PVGTGLNIVHTAVRTFGGTVTVADSDLGGTAFEVALPRDAE 397 KinA1 ADCTDDNTATIHVADNGPGVPDAQKDAIFGKGETGLDSDGTGMGLYLVDTLVDSVGGRVTVSDNDPTGAVFSVTLRTGP- 606

Figure 3.15 Alignments of the N- and C-terminal sequences of OE2712R with the N-terminal sequences of MpcT (Htr14) and its putative ortholog from N. pharaonis, and with the C-terminal sequence of KinA, respectively.

Residues which are identical and similar in at least two of the aligned sequences are highlighted black and grey, respectively. (A) Brackets indicate the beginning and end of the region that is well conserved in Htr14 from H. salinarum and from N. pharaonis as well as in OE2712R (comprising residues W12 - L166 of Htr14 from H. salinarum). Indicated are the predicted transmembrane regions (t) interrupted by a hairpin (h), and the positions of the two GDL-motifs (gdl) indicative of HAMP domains (of which the second one is missing in OE2712R). An asterisk indicates the position of the putatively phosphorylatable His203 located within a sequence of OE2712R (italicized) which shows significant homology to the putative histidine kinase KinA but not to Htr14. (B) Residues 170 - 397 of OE2712R (414 residues) are aligned to residues 369 - 606 of KinA (606 residues). The position of His203 from OE2712R is marked by an asterisk. The sequence of OE2712R, which shows significant homology to Htr14 but not to KinA, is italicized.

deletion of this gene in strain MKK101 had no detectable effect on BR-dependent phototaxis (data not shown), which excludes it as being a partner with MpcT in ∆Ψ sensing.

Although a role for MpcT as the direct sensor of changes in ∆Ψ is supported by the absence of a cotranscribed receptor gene, it cannot yet be ruled out that one or more additional proteins are required to interact with MpcT in the process of ∆∆Ψ sensing. However, contrary to signaling via Htr1 and Htr2, which in both cases requires a physical interaction with the corresponding photoreceptors, such an interaction of MpcT with BR or HR is not assumed. A ∆∆Ψ, generated via BR or HR, should be detectable anywhere along the cell membrane, and does not necessarily require a protein-protein interaction between the sensor and the generator of ∆∆Ψ. As photostimuli can be sensed in both combinations, BR/MpcT or HR/MpcT, a physical interaction would require that MpcT interact alternatively with two different receptors, i.e. with BR as well as with HR. Taking into account the differences especially in the structures of the membrane- exposed surfaces, the tilt of the helices and the photocycles of HR and BR, this seems rather unlikely.