Alternative Model for RND-Type Efflux Pump

RND (resistance-nodulation-division) transporters are found in several Gram-negative bacteria species. These proteins form a complex along with membrane fusion proteins and outer membrane factors. This complex acts as an efflux pump, transporting several different molecules directly from the cytoplasm to the extracellular medium. Although the crystallographic structure of each protein of the complex is known, the complete complex assembly is not yet fully characterized. In 2014, a model for the complete system, based upon a cryoelectron-microscopy map, was proposed. In the present work, we propose an alternative model that also satisfies the volume obtained from the cryoelectron-microscopy assay. In this model, the AcrA helical domains are interleaved to the helical domains of the TolC protein, increasing the diameter of the formed pore. We believe that this model represents a better model for RND-type efflux pump and might contribute to the characterization of this system.

Keywords:
RND efflux system; comparative modeling; efflux pump; molecular modeling; protein structure

Authorship

Pedro Henrique Monteiro Torres ^**e-mail: monteirotorres@gmail.com

Diretoria de Metrologia Aplicada às Ciências da Vida, Instituto Nacional de Metrologia, Qualidade e Tecnologia, 25250-020 Duque de Caxias-RJ, BrazilInstituto Nacional de Metrologia, Qualidade e TecnologiaBrazilDuque de Caxias, RJ, BrazilDiretoria de Metrologia Aplicada às Ciências da Vida, Instituto Nacional de Metrologia, Qualidade e Tecnologia, 25250-020 Duque de Caxias-RJ, Brazil

Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, 21044-020 Rio de Janeiro-RJ, BrazilUniversidade Federal do Rio de JaneiroBrazilRio de Janeiro, RJ, BrazilInstituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, 21044-020 Rio de Janeiro-RJ, Brazil

Pedro Geraldo Pascutti

Paulo Mascarello Bisch

Manuela Leal da Silva

*e-mail: monteirotorres@gmail.com

SCIMAGO INSTITUTIONS RANKINGS

Figures | Tables

Figures (4)
Tables (1)

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Figure 1
(a) Schematic representation of the alignment procedure. The colored bars represent the sequences retrieved from UniProt:P31224 (AcrB) in red, B6I0B3 (AcrA) in green and P02930 (TolC) in yellow. The black segments over dashed lines represent the regions of the sequence that were employed in the alignment procedure. The aligned segments spans are shown under the colored bars; (b) cartoon representation of each template structure, with boxes highlighting the regions that were used as templates. The red box consists of the template used to model AcrB sequence. The set of green boxes represents the regions used to model the AcrA sequence and the set of yellow boxes are the regions used to model TolC sequence. The black box, highlights the new model for the interface region between AcrA and TolC; (c) whole constructed model in cartoon and surface representations. The TolC trimer is colored in yellow, the AcrA hexameric assembly is colored in dark and light green (identical sequences) and the AcrB trimer in red. The gray bars represent the polar groups of the inner membrane (IM) and outer membrane (OM), the space delimited between the central bars depicts the periplasmic space (PS).

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Figure 2
Ramachandran plot: 88.9% of the residues are in the most favored region (cyan) 9.4% are in the allowed region (light blue), 0.8% in the generously allowed region (gray) and 0.9% in disallowed regions. Black squares correspond to non-glycine residues inside the permitted regions, while red squares correspond to the non-glycine residues outside the permitted regions. Black triangles correspond to glycine residues.

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Figure 3
Dope scores and G-factors calculated for 100 generated models. The model displayed in Figures 1 and 4 corresponds to the upper left point.

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Figure 4
(a) Contact interface between the TolC protomers (in yellow) and the AcrA protomers (in green). The hydrophobic residues that form this interface are highlighted in a sphere representation. Residues that belong to the TolC protomers are colored in dark grey, whilst residues that belong to the AcrA protomers are colored in white. The interface region was modeled using the crystallographic structure of MacA (PDB code 4DK0), which consists of a homododecameric assembly presenting a tip-to-tip interaction between the helical domains; (b) superposition of the TolC trimers: the crystallographic structure under PDB ID 2XMN (represented as grey surface) and the new proposed model, colored by chain (in green, magenta and cyan spheres). The pore diameter was measured as the distance from the β-carbon of V370 from chains A (green) and B (cyan); (c) complete structure of AcrB-AcrA-TolC complex superimposed to the Cryo-EM map volume. The AcrB are colored in red, AcrA in green and TolC in yellow. The tip-to-tip arrangement between AcrA and TolC helical domains is compatible with the bulge in the Cryo-EM map, located at the interface region. This superimposition was achieved using the Chimera software; (d) schematic representation of the different spatial arrangement of the helical domains of AcrA (green) and TolC (yellow). On the left, the representation of the model proposed by Du et al.⁶6 Du, D.; Wang, Z.; James, N. R.; Voss, J. E.; Klimont, E.; Ohene-Agyei, T.; Venter, H.; Chiu, W.; Luisi, B. F.; Nature 2014, 509, 512. and on the right the model proposed in this work.

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Table 1
Identity, similarity and coverage values for the performed alignments

Figure 1 (a) Schematic representation of the alignment procedure. The colored bars represent the sequences retrieved from UniProt:P31224 (AcrB) in red, B6I0B3 (AcrA) in green and P02930 (TolC) in yellow. The black segments over dashed lines represent the regions of the sequence that were employed in the alignment procedure. The aligned segments spans are shown under the colored bars; (b) cartoon representation of each template structure, with boxes highlighting the regions that were used as templates. The red box consists of the template used to model AcrB sequence. The set of green boxes represents the regions used to model the AcrA sequence and the set of yellow boxes are the regions used to model TolC sequence. The black box, highlights the new model for the interface region between AcrA and TolC; (c) whole constructed model in cartoon and surface representations. The TolC trimer is colored in yellow, the AcrA hexameric assembly is colored in dark and light green (identical sequences) and the AcrB trimer in red. The gray bars represent the polar groups of the inner membrane (IM) and outer membrane (OM), the space delimited between the central bars depicts the periplasmic space (PS).

Figure 2 Ramachandran plot: 88.9% of the residues are in the most favored region (cyan) 9.4% are in the allowed region (light blue), 0.8% in the generously allowed region (gray) and 0.9% in disallowed regions. Black squares correspond to non-glycine residues inside the permitted regions, while red squares correspond to the non-glycine residues outside the permitted regions. Black triangles correspond to glycine residues.

Figure 3 Dope scores and G-factors calculated for 100 generated models. The model displayed in Figures 1 and 4 corresponds to the upper left point.

Figure 4 (a) Contact interface between the TolC protomers (in yellow) and the AcrA protomers (in green). The hydrophobic residues that form this interface are highlighted in a sphere representation. Residues that belong to the TolC protomers are colored in dark grey, whilst residues that belong to the AcrA protomers are colored in white. The interface region was modeled using the crystallographic structure of MacA (PDB code 4DK0), which consists of a homododecameric assembly presenting a tip-to-tip interaction between the helical domains; (b) superposition of the TolC trimers: the crystallographic structure under PDB ID 2XMN (represented as grey surface) and the new proposed model, colored by chain (in green, magenta and cyan spheres). The pore diameter was measured as the distance from the β-carbon of V370 from chains A (green) and B (cyan); (c) complete structure of AcrB-AcrA-TolC complex superimposed to the Cryo-EM map volume. The AcrB are colored in red, AcrA in green and TolC in yellow. The tip-to-tip arrangement between AcrA and TolC helical domains is compatible with the bulge in the Cryo-EM map, located at the interface region. This superimposition was achieved using the Chimera software; (d) schematic representation of the different spatial arrangement of the helical domains of AcrA (green) and TolC (yellow). On the left, the representation of the model proposed by Du et al.⁶6 Du, D.; Wang, Z.; James, N. R.; Voss, J. E.; Klimont, E.; Ohene-Agyei, T.; Venter, H.; Chiu, W.; Luisi, B. F.; Nature 2014, 509, 512. and on the right the model proposed in this work.

Table 1 Identity, similarity and coverage values for the performed alignments

Template	AcrB			AcrA			TolC
Template	I / %	S / %	C / %	I / %	S / %	C / %	I / %	S / %	C / %
4CDI	99	99	98
2F1M				91	91	61
2XMN							99	99	100
4DK0				28	41	76	35	43	9

I: identity; S: similarity; C: coverage.

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[1] *e-mail: monteirotorres@gmail.com