Protein target highlights in CASP15: Analysis of models by structure providers

We present an in-depth analysis of selected CASP15 targets, focusing on their biological and functional significance. The authors of the structures identify and discuss key protein features and evaluate how effectively these aspects were captured in the submitted predictions. While the overall ability to predict three-dimensional protein structures continues to impress, reproducing uncommon features not previously observed in experimental structures is still a challenge. Furthermore, instances with conformational flexibility and large multimeric complexes highlight the need for novel scoring strategies to better emphasize biologically relevant structural regions. Looking ahead, closer integration of computational and experimental techniques will play a key role in determining the next challenges to be unraveled in the field of structural molecular biology

Recognition of the Type III Secretion Substrate YscX by the Export Gate Protein YscV

Gilzer D. Recognition of the Type III Secretion Substrate YscX by the Export Gate Protein YscV. Bielefeld: Universität Bielefeld; 2023

Direct interaction of a chaperone-bound type III secretion substrate with the export gate

Gilzer D, Schreiner M, Niemann H. Direct interaction of a chaperone-bound type III secretion substrate with the export gate. Nature Communications . 2022;13(1): 2858.Several gram-negative bacteria employ type III secretion systems (T3SS) to inject effector proteins into eukaryotic host cells directly from the bacterial cytoplasm. The export gate SctV (YscV in Yersinia) binds substrate:chaperone complexes such as YscX:YscY, which are essential for formation of a functional T3SS. Here, we present structures of the YscX:YscY complex alone and bound to nonameric YscV. YscX binds its chaperone YscY at two distinct sites, resembling the heterotrimeric complex of the T3SS needle subunit with its chaperone and co-chaperone. In the ternary complex the YscX N-terminus, which mediates YscX secretion, occupies a binding site within one YscV that is also used by flagellar chaperones, suggesting the interaction's importance for substrate recognition. The YscX C-terminus inserts between protomers of the YscV ring where the stalk protein binds to couple YscV to the T3SS ATPase. This primary YscV-YscX interaction is essential for the formation of a secretion-competent T3SS. © 2022. The Author(s)

Direct interaction of a chaperone-bound type III secretion substrate with the export gate

Several gram-negative bacteria employ type III secretion systems (T3SS) to inject effector proteins into eukaryotic host cells directly from the bacterial cytoplasm. The export gate SctV (YscV in Yersinia) binds substrate:chaperone complexes such as YscX:YscY, which are essential for formation of a functional T3SS. Here, we present structures of the YscX:YscY complex alone and bound to nonameric YscV. YscX binds its chaperone YscY at two distinct sites, resembling the heterotrimeric complex of the T3SS needle subunit with its chaperone and co-chaperone. In the ternary complex the YscX N-terminus, which mediates YscX secretion, occupies a binding site within one YscV that is also used by flagellar chaperones, suggesting the interaction’s importance for substrate recognition. The YscX C-terminus inserts between protomers of the YscV ring where the stalk protein binds to couple YscV to the T3SS ATPase. This primary YscV–YscX interaction is essential for the formation of a secretion-competent T3SS

The type III secretion chaperone SctY may shield the hydrophobic export gate-binding C-terminus of its substrate SctX

Gilzer D, Kowal J, Flottmann F, Niemann H. The type III secretion chaperone SctY may shield the hydrophobic export gate-binding C-terminus of its substrate SctX. Acta Crystallographica Section D : Structural Biology . 2023;D79(6):508-517.Gram-negative bacteria such as Aeromonas and Yersinia spp. have developed mechanisms to inhibit the immune defense of their host. Effector proteins are directly injected into the host cytoplasm from the bacterial cytosol via type III secretion systems (T3SSs), where they modulate the cytoskeleton and signaling of the cell. Assembly of, and secretion via, T3SSs is tightly regulated by a number of bacterial proteins, including SctX (AscX in Aeromonas), the secretion of which is essential for T3SS function. Here, crystal structures of AscX in complex with SctY chaperones from Yersinia or Photorhabdus spp. carrying homologous T3SSs are described. There are crystal pathologies in all cases, with one crystal form diffracting anisotropically and the other two exhibiting strong pseudotranslation. The new structures reveal that the positioning of the substrate is very similar on different chaperones. However, the two C-terminal SctX helices that cap the N-terminal tetratricopeptide repeat of SctY shift and tilt depending on the identity of the chaperone. Moreover, the C-terminus of the alpha3 helix of AscX exhibits an unprecedented kink in two of the structures. In previous structures, the C-terminus of SctX protrudes beyond the chaperone as a straight helix: a conformation that is required for binding to the nonameric export gate SctV but that is unfavorable for binary SctX-SctY complexes due to the hydrophobicity of helix alpha3 of SctX. A kink in helix alpha3 may allow the chaperone to shield the hydrophobic C-terminus of SctX in solution. open access

Crystals of SctV from different species reveal variable symmetry for the cytosolic domain of the type III secretion system export gate

Type III secretion systems (T3SSs) are proteinaceous devices employed by Gram-negative bacteria to directly transport proteins into a host cell. Substrate recognition and secretion are strictly regulated by the export apparatus of the so-called injectisome. The export gate SctV engages chaperone-bound substrates of the T3SS in its nonameric cytoplasmic domain. Here, the purification and crystallization of the cytoplasmic domains of SctV from Photorhabdus luminescens (LscV$_C$) and Aeromonas hydrophila (AscV$_C$) are reported. Self-rotation functions revealed that LscV$_C$ forms oligomers with either eightfold or ninefold symmetry in two different crystal forms. Similarly, AscV$_C$ was found to exhibit tenfold rotational symmetry. These are the first instances of SctV proteins forming non-nonameric oligomers

Crystals of SctV from different species reveal variable symmetry for the cytosolic domain of the type III secretion system export gate

Gilzer D, Baum E, Lieske N, Kowal J, Niemann H. Crystals of SctV from different species reveal variable symmetry for the cytosolic domain of the type III secretion system export gate. Acta Crystallographica Section F : Structural Biology Communications . 2022;78(11):386-394.Type III secretion systems (T3SSs) are proteinaceous devices employed by Gram-negative bacteria to directly transport proteins into a host cell. Substrate recognition and secretion are strictly regulated by the export apparatus of the so-called injectisome. The export gate SctV engages chaperone-bound substrates of the T3SS in its nonameric cytoplasmic domain. Here, the purification and crystallization of the cytoplasmic domains of SctV from Photorhabdus luminescens (LscVC) and Aeromonas hydrophila (AscVC) are reported. Self-rotation functions revealed that LscVC forms oligomers with either eightfold or ninefold symmetry in two different crystal forms. Similarly, AscVC was found to exhibit tenfold rotational symmetry. These are the first instances of SctV proteins forming non-nonameric oligomers

Biphasic Formation of 2D Nanomembranes by Photopolymerization of Diacetylene Lipids as Revealed by Infrared Difference Spectroscopy

Gilzer D, Hillmann R, Gött-Zink L, et al. Biphasic Formation of 2D Nanomembranes by Photopolymerization of Diacetylene Lipids as Revealed by Infrared Difference Spectroscopy. Langmuir. 2019;35(29):9343-9351

Preparation of 2D Phospholipid and Copolymer Nanomembranes

Hillmann R, Gilzer D, Knust S, et al. Preparation of 2D Phospholipid and Copolymer Nanomembranes. Materials Today: Proceedings. 2017;4:S87-S92

Characterization of Robust and Free-Standing 2D-Nanomembranes of UV-Polymerized Diacetylene Lipids

Hillmann R, Viefhues M, Goett-Zink L, et al. Characterization of Robust and Free-Standing 2D-Nanomembranes of UV-Polymerized Diacetylene Lipids. Langmuir. 2018;34(10):3256-3263