Interdomain signalling in the blue-light sensing and GTP-binding protein YtvA: A mutagenesis study uncovering the importance of specific protein sites

Abstract

YtvA from Bacillus subtilis is a blue-light responsive, flavin-binding photoreceptor, built of a light-sensing LOV domain (aa 25-126) and an NTP (nucleoside triphosphate)-binding STAS domain (aa 147-261). The STAS domain is supposed to be the effector part of the protein or a secondary switch. Both domains are connected by a linker polypeptide. The active form of YtvA is generated upon light excitation, causing the formation of a covalent bond between a cysteine residue (Cys62) in the LOV domain and the position 4a of the flavin chromophore. This photoadduct formation within the LOV domain results in a conformational change of the NTP-binding cavity, evidencing intra-protein signal transmission. We have previously shown that Glu105, localized on the beta-scaffold of the LOV-core, is involved in this process. Here, we extend this work by the identification of further residues that upon mutation supress or strongly impair signal transmission by interfering with the communication between the two domains. These comprise L106 and D109 on the LOV domain; K130 and K134 on the linker region; D193, L194 and G196 within the DLSG GTP-binding motif (switch region) and N201 on the STAS domain. Furthermore in the mutated S195A and D193A proteins, GTP affinity is diminished. Other mutations investigated have little or no effect on signal transmission and GTP-binding affinity: R63K that was found to accelerate the thermal recovery of the parent state ca. ten-fold; K128A, Q129A and Y132A within the linker region, and S183A and S212A on the STAS domain. The results show a key role of the LOV domain beta-scaffold and of positively charged residues within the linker for intra-protein signal transmission. Furthermore they evidence the conformational switch function of a structurally conserved strand-loop-helix region (bearing the DLSG GTP-binding motif and N201) within the STAS domain that constitutes a novel GTP-binding fold.

Solution Structure of the Guanine Nucleotide-binding STAS Domain of SLC26-related SulP Protein Rv1739c from Mycobacterium tuberculosis

“…S4F), a property shared with SpoIIAA (22). The presence of 1 mM Mg 2ϩ reduced binding affinity 2-fold for GTP and ϳ4-fold for GDP (Table 2), as noted for the STAS domain of B. subtilis photosensor YtvA (48). These data are the first to demonstrate guanine nucleotide binding to a SulP transporter STAS domain.…”

“…B. subtilis SpoIIAA exhibits GTPase activity (22), but the GTP-binding protein YtvA lacks detectable GTPase activity (48). We therefore evaluated Rv1739c for the presence of intrinsic nucleotidase activity.…”

“…S7A). Some of the nucleotide-perturbed residues, including those of the N-terminal region, Val 48 and Val 61 , correspond to regions in anti-factor antagonist TM1081, which exhibit millisecond scale conformational changes in solution but not in the crystal, suggesting their involvement in binding or catalytic activity (27).…”

LOV and BLUF flavoproteins’ regulatory photoreceptors of microorganisms and photosensory actuators in optogenetic systems

“…Кроме того, фотоактивиро-ванный YtvA in vivo действует как положительный регулятор транскрипционного фактора общего стресса σB [23]. Поскольку мутации, нарушающие связывание ГТФ в STAS-домене или междоменное распространение светоиндуцированного сигнала в фоторецепторе, подавляют активированную синим светом σB-зависимую транскрипцию in vivo, счита-ется, что связывание ГТФ необходимо для прояв-ления функциональной активности YtvA [23][24][25].…”

“…Исследования структурных изменений, про-исходящих в LOV-домене YtvA, который соединен с эффекторным STAS-доменом Jα-спиралью, по-казали, что, в отличие от таковых у фототропинов, они не затрагивают деструктуризацию Jα-спирали [24,26]. Это связано с различиями в четвертичной структуре и ориентации Jα, которая у YtvA-LOV-домена образует "coiled-coil"-конфигурацию [23].…”