Dissection of the ATPase active site of McdA reveals the sequential steps essential for carboxysome distribution

Hakim, Pusparanee; Hoang, Y; Vecchiarelli, Anthony G.

doi:10.1091/mbc.e21-03-0151

Cited by 5 publications

(4 citation statements)

References 76 publications

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“…We began by expressing individual HO shell proteins (HO BMC-H, HO BMC-T1, and HO BMC-P) and assaying whether they localized to carboxysomes and/or perturbed carboxysome morphology, function, or positioning. To monitor the localization and morphology of carboxysomes, we created a carboxysome reporter background by genomically-integrating an additional copy of the small subunit of Rubisco (RbcS) fused at the c-terminus to mTurquoise2 (mTQ) under a second copy of the native rbcS promoter, as has been characterized previously (MacCready et al, 2018; Hakim et al, 2021; Rillema et al, 2021) . Simultaneously, HO BMC-H, HO BMC-T1, or HO BMC-P fluorescently fused at the c-terminus to mNeonGreen (mNG) and expressed using a synthetic riboswitch was inserted upstream of the carboxysome reporter.…”

Section: Resultsmentioning

confidence: 99%

Orthogonality of shell proteins across BMC subclasses in cyanobacteria

MacCready,

Dwyer,

Kerfeld

et al. 2024

Preprint

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Bacterial microcompartments (BMC) are protein-based organelles broadly distributed across all bacterial phyla and subclassified into ≥60 functional variants. Despite their evolutionary and metabolic diversity, shell proteins that structurally compose the BMC surface are closely related across BMC classes. Herein, we sought to identify molecular and physiological features that could promote independent operation of more than one BMC type within the same cell by reducing inter-organelle cross-talk of shell proteins. We heterologously expressed shell proteins from the structurally well-defined BMC ofHaliangium ochraceum(HO) withinSynechococcus elongatusPCC 7942, a model cyanobacterium containing the β-carboxysome. We find considerable cross-reactivity of the HO hexameric shell protein (HO BMC-H) with components of the β-carboxysome; HO BMC-H can integrate into carboxysomes, disrupt its ultrastructural organization, and impair its associated CO2fixation reactions.S. elongatusis unable to maintain the integrity of the β-carboxysome over time when HO BMC-H is expressed in the absence of one or more of three broad strategies that act to increase the orthogonality between HO and carboxysome BMC shell proteins: i) reduced expression of promiscuous shell proteins; ii) sequestration of free HO BMC-H proteins via co-expression of other members of the same HO shell protein class, or; iii) heterologous expression of BMC positional system proteins McdAB (Maintenance ofcarboxysomedistribution AB), revealing a putative moonlighting function of the McdAB protein family. Our results have implications for bacteria that encode more than one BMC within their genome and may have translational implications for the use of engineered BMCs for biotechnological applications.

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Section: Resultsmentioning

confidence: 99%

Orthogonality of shell proteins across BMC subclasses in cyanobacteria

MacCready,

Dwyer,

Kerfeld

et al. 2024

Preprint

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“…McdB functions as an adaptor protein for the carboxysome positioning ATPase, McdA, which is a member of the ParA/MinD-family of positioning ATPases (13, 14, 19). This family is widespread in bacteria, and is responsible for spatially regulating a variety of genetic- and protein-based cargos (13, 14).…”

Section: Discussionmentioning

confidence: 99%

“…Despite their diversity, α- and β-McdB proteins have been shown to form condensates in vitro (11, 12, 18). The processes underlying condensate formation in vitro can influence subcellular organization in vivo in both eukaryotes and prokaryotes (19, 20). Along these lines, we recently found evidence suggesting that condensate formation by Se McdB may play a role in its association with carboxysomes in vivo (21).…”

Section: Introductionmentioning

confidence: 99%

An invariant C-terminal tryptophan in McdB mediates its interaction and positioning function with carboxysomes

Basalla,

Ghalmi,

Hoang

et al. 2023

Preprint

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Bacterial microcompartments (BMCs) are widespread, protein-based organelles that regulate metabolism. The model for studying BMCs is the carboxysome, which facilitates carbon-fixation in several autotrophic bacteria. Carboxysomes can be distinguished as type α or ß, which are structurally and phyletically distinct. We recently characterized the Maintenance of Carboxysome Distribution (Mcd) systems responsible for spatially regulating α- and ß-carboxysomes, consisting of the proteins McdA and McdB. McdA is an ATPase that drives carboxysome positioning, and McdB is the adaptor protein that directly interacts with carboxysomes to provide cargo specificity. The molecular features of McdB proteins that specify their interactions with carboxysomes, and whether these are similar between α- and ß-carboxysomes, remain unknown. Here, we identify C-terminal motifs containing an invariant tryptophan necessary for α- and ß-McdBs to associate with α- and ß-carboxysomes, respectively. Substituting this tryptophan with other aromatic residues reveals corresponding gradients of carboxysome colocalization and positioning by McdBin vivo. Intriguingly, these gradients also correlate with the ability of McdB to form condensatesin vitro. The results reveal a shared mechanism underlying McdB adaptor protein binding to carboxysomes, and potentially other BMCs. Our findings also implicate condensate formation as playing a key role in this association.SIGNIFICANCE STATEMENTMaintenance of carboxysome distribution protein B (McdB) is necessary for positioning a widespread class of protein-based organelles in bacteria that regulate metabolism. Without McdB, these organelles aggregate and lose functionality. How McdB interacts with and positions these organelles is unknown.We determine that an invariant tryptophan is necessary for McdB to interact with and position its organelle. A similar mechanism occurs in two diverse bacterial cell types, both relying on the invariant tryptophan.This class of bacterial organelle includes compartments involved in bacterial pathogenesis and carbon fixation. Our results therefore advance our understanding and applications of these organelles.

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“…The interplay between McdA gradients on the nucleoid and McdB-bound carboxysomes result in the equal spacing of carboxysomes down the cell length of rod-shaped bacteria. This mode of spatial regulation by McdA is typical for the widespread and well-studied ParA/MinD family of positioning ATPases, of which McdA is a member ( Hakim et al, 2021 ). ParA/MinD ATPases spatially organize an array of genetic- and protein-based cargos in the cell, including plasmids, chromosomes, the divisome, flagella, and other mesoscale complexes ( Lutkenhaus, 2012 ; Vecchiarelli et al, 2012 ).…”

Section: Introductionmentioning

confidence: 88%

Dissecting the phase separation and oligomerization activities of the carboxysome positioning protein McdB

Basalla,

Mak,

Byrne

et al. 2023

eLife

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Across bacteria, protein-based organelles called bacterial microcompartments (BMCs) encapsulate key enzymes to regulate their activities. The model BMC is the carboxysome that encapsulates enzymes for CO2 fixation to increase efficiency and is found in many autotrophic bacteria, such as cyanobacteria. Despite their importance in the global carbon cycle, little is known about how carboxysomes are spatially regulated. We recently identified the two-factor system required for the maintenance of carboxysome distribution (McdAB). McdA drives the equal spacing of carboxysomes via interactions with McdB, which associates with carboxysomes. McdA is a ParA/MinD ATPase, a protein family well-studied in positioning diverse cellular structures in bacteria. However, the adaptor proteins like McdB that connect these ATPases to their cargos are extremely diverse. In fact, McdB represents a completely unstudied class of proteins. Despite the diversity, many adaptor proteins undergo phase separation, but functional roles remain unclear. Here, we define the domain architecture of McdB from the model cyanobacterium Synechococcus elongatus PCC 7942, and dissect its mode of biomolecular condensate formation. We identify an N-terminal intrinsically disordered region (IDR) that modulates condensate solubility, a central coiled-coil dimerizing domain that drives condensate formation, and a C-terminal domain that trimerizes McdB dimers and provides increased valency for condensate formation. We then identify critical basic residues in the IDR, which we mutate to glutamines to solubilize condensates. Finally, we find that a condensate-defective mutant of McdB has altered association with carboxysomes and influences carboxysome enzyme content. The results have broad implications for understanding spatial organization of BMCs and the molecular grammar of protein condensates.

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Dissection of the ATPase active site of McdA reveals the sequential steps essential for carboxysome distribution

Cited by 5 publications

References 76 publications

Orthogonality of shell proteins across BMC subclasses in cyanobacteria

Orthogonality of shell proteins across BMC subclasses in cyanobacteria

An invariant C-terminal tryptophan in McdB mediates its interaction and positioning function with carboxysomes

Dissecting the phase separation and oligomerization activities of the carboxysome positioning protein McdB

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