Biology subjects

Marquez, M. D.

Publications and source records attributed to Marquez, M. D..

5 recordsLinked to original sources

bioRxiv2026

Nar1 binds the cytosolic iron sulfur cluster assembly targeting complex via a bipartite interaction interface

The cytosolic iron-sulfur cluster assembly (CIA) pathway maturates essential nuclear and cytosolic Fe-S proteins required for genome maintenance and cellular metabolism. Nar1 (also called CIAO3 or IOP1) is a conserved Fe-S protein that connects the early and late steps of the CIA pathway, yet the molecular basis for its proposed function as a metallocluster carrier remains poorly defined. In particular, the interactions responsible for Nar1 recruitment to the CIA targeting complex (CTC) during cluster delivery remain unknown. Here, we define the molecular basis for Nar1 recruitment to the CTC using biochemical reconstitution, quantitative protein-protein interaction assays, and AlphaFold modeling. Our data reveal that Nar1 binds the CTC through two distinct interfaces. A primary interface comprises an electrostatic interaction that anchors Nar1 to a conserved acidic surface on the Cia1 subunit of the CTC and a secondary interface involves binding of Nar1s divergent targeting complex recognition peptide at the Cia1-Cia2 interface. Thus, Nar1 engages a conserved CTC surface that serves as a recruitment platform for multiple binding partners, including CIA clients. Computational structural models position the putative Fe-S cluster donor site of Nar1 adjacent to a proposed acceptor site on Cia2, suggesting that this bipartite binding mechanism positions Nar1 for transfer of an Fe-S cluster to the targeting complex. Together, these findings resolve conflicting models for Nar1 recruitment and establish a mechanistic framework for understanding how the CTC engages multiple binding partners during cytosolic iron-sulfur protein maturation.

biochemistry↗

bioRxiv2025

Structures of folding intermediates on BAM show diverse substrates fold by a uniform mechanism

The outer membranes of mitochondria, chloroplasts, and Gram-negative bacteria contain {beta}-barrel membrane proteins that are assembled by conserved multi-subunit machines. In bacteria, the {beta}-barrel assembly machine (BAM) folds over a hundred compositionally different substrates into barrels that vary greatly in size. Some larger barrels require globular proteins to plug the barrel lumen. How a single machine can assemble such different barrels is unknown. Here we report three structures representing progressively folded stages of a 16-stranded barrel engaged with BAM, as well as the structure of a late-stage folding intermediate of a 26-stranded substrate folding around its soluble lipoprotein plug on BAM. We find that BAM catalyzes folding of these substrates by a uniform mechanism in which BAM undergoes major distortions to accommodate the nascent barrel.

biochemistry↗

bioRxiv2025

The Cia1 and Cia2 subunits of the CTC mediate recognition of apo-FeS proteins with a C-terminal targeting complex recognition motif

The cytosolic iron-sulfur cluster assembly (CIA) targeting complex is responsible for maturation of cytosolic and nuclear iron-sulfur enzymes, numbering >30 proteins critical for fundamental processes such as DNA replication and repair. Up to 25% of these client proteins terminate in a targeting complex recognition (TCR) motif. This carboxy-terminal tripeptide motif recruits the CIA targeting complex (CTC) to the client so that the metallocluster can be inserted. Herein, we use a combination of computational, biochemical and biophysical approaches to determine that the clients bearing a TCR motif docks at the interface of the Cia1 and Cia2 subunits of the CTC. Thus, mutations destabilizing the Cia1-Cia2 complex also disrupt TCR-based client identification by the CTC. Our study also reveals that the understudied human Cia2 paralog CIAO2A, which is proposed to be a specific targeting factor for iron regulatory protein 1, can recruit clients terminating in the TCR peptide. These data signal that CIAO2A plays a more general role in iron-sulfur protein maturation than previously appreciated. Taken together, our findings deepen our understanding of the molecular basis for client recognition by the CTC that is critical to understand the impact of CIA function in human health and disease.

biochemistry↗

bioRxiv2024

Native β-barrel substrates pass through two shared intermediates during folding on the BAM complex

The assembly of {beta}-barrel proteins into membranes is mediated by the evolutionarily conserved BAM complex. In Escherichia coli, BAM folds numerous substrates which vary considerably in size and shape. How BAM is able to efficiently fold such a diverse array of {beta}-barrel substrates is not clear. Here, we develop a disulfide crosslinking method to trap native substrates in vivo as they fold on BAM. By placing a cysteine within the luminal wall of the BamA barrel as well as in the substrate {beta}-strands, we can compare the residence time of each substrate strand within the BamA lumen. We validated this method using two defective, slow-folding substrates. We used this method to characterize stable intermediates which occur during folding of two structurally different native substrates. Strikingly, these intermediates occur during identical stages of folding for both substrates: soon after folding has begun, and just before folding is completed. We suggest that these intermediates arise due to barriers to folding that are common between {beta}-barrel substrates, and that the BAM catalyst is able to fold so many different substrates because it addresses these common challenges. Significance StatementThe outer membrane of Gram-negative bacteria is a barrier which protects these organisms from many antimicrobial agents. Here, we study the machine responsible for folding and inserting integral {beta}-barrel proteins into the membrane: BAM. Outer membrane integrity and cell viability is dependent on the proper function of BAM. Here we show that stable intermediates exist on the folding pathway of native substrates. We also show that mutant substrates that increase the stability of these native intermediates can stall during folding. This creates permeability defects that can be exploited by antibiotics that normally do not cross the outer membrane. These observations could enable the design of strategies to combat Gram-negative pathogens.

biochemistry↗

bioRxiv2023

Cytosolic iron-sulfur protein assembly system identifies clients by a C-terminal tripeptide

The eukaryotic cytosolic Fe-S protein assembly (CIA) machinery inserts iron-sulfur (Fe-S) clusters into cytosolic and nuclear proteins. In the final maturation step, the Fe-S cluster is transferred to the apo-proteins by the CIA-targeting complex (CTC). However, the molecular recognition determinants of client proteins are unknown. We show that a conserved [LIM]-[DES]-[WF]-COO- tripeptide present at the C-terminus of clients is necessary and sufficient for binding to the CTC in vitro and directing Fe-S cluster delivery in vivo. Remarkably, fusion of this TCR (target complex recognition) signal enables engineering of cluster maturation on a non-native protein via recruitment of the CIA machinery. Our study significantly advances our understanding of Fe-S protein maturation and paves the way for bioengineering applications. One-Sentence SummaryA C-terminal tripeptide guides eukaryotic iron-sulfur cluster insertion into cytosolic and nuclear proteins.

biochemistry↗