About

Professor Marcelo C. Sousa is a researcher at the University of Colorado Boulder, leading the Sousa Research Group within the College of Arts and Sciences. His research program focuses on understanding fundamental cellular processes at the membrane interface at a molecular level, utilizing a multidisciplinary approach that includes X-ray crystallography, electron microscopy, and complementary biochemical and biophysical techniques such as single molecule studies. His projects encompass the structural and mechanistic analysis of enzymes involved in Lipid-A modification related to antibiotic resistance, the molecular mechanisms of folding and insertion of membrane proteins in bacterial and mitochondrial outer membranes, and the determinants of bacterial protein secretion. His work aims to elucidate the structures and functions of key membrane-associated proteins and complexes, contributing to the development of targeted inhibitors and advancing knowledge of membrane biology.

Research topics

• Chemistry
• Biochemistry
• Biology
• Stereochemistry
• Cell biology

Selected publications

• A fluorescent reporter and single-turnover kinetics reveal new insight into BAM complex function

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-05-31

  preprintOpen accessSenior authorCorresponding

  Abstract The β-barrel assembly machine (BAM) is an essential protein complex that folds and inserts outer membrane β-barrel proteins (OMPs) into the bacterial outer membrane. The BAM complex contains the essential BamA OMP with five soluble polypeptide transport-associated (POTRA) domains, which scaffold the essential BamD lipoprotein and the non-essential lipoproteins BamB/C/E. The importance of each BAM component has been investigated primarily using cell-based phenotypic assays, and structural data have revealed insights into the role of the BamA β-barrel in OMP folding. However, in vitro quantitative analysis for the function of each BAM component has been challenging. We describe the development of a fluorescent reporter of OMP folding, tOmpA-A488, which we use to obtain single-turnover kinetic parameters for wildtype BAM complex in vitro . We observe a k fold of 0.78 ± 0.15 min −1 and approximate substrate affinity of 3.1 ± 1.1 µM consistent with estimates of in vivo requirements. We also find that, contrary to prevailing models, POTRA domain deletions that include POTRA3, which is essential in cells, do not drastically impact activity. This indicates that the first three POTRA domains do not play a major role in binding or folding OMPs under single-turnover conditions, suggesting a different role in cells. Furthermore, we find that BamA alone is inactive in E. coli lipid liposomes, and the gain-of-function mutant BamA E470K does not rescue activity in vitro . The single-turnover kinetics enabled by the fluorescent reporter presented here defines a robust platform for quantitative evaluation of the folding activity of wildtype and mutant BAM complexes. Significance The folding of outer membrane proteins (OMPs) in Gram-negative bacteria requires the essential β-barrel assembly machine (BAM). By developing a fluorescent OMP folding reporter, we have unlocked insight into BAM activity in vitro , opening the door for rigorous evaluation of BAM mutants and putative inhibitors. We also discovered that, contrary to current models, the BamA POTRA1-3 domains do not contribute significantly to catalysis, despite being essential for bacterial growth. We propose that, consistent with biochemical, structural, and live cell imaging, the in vivo role of BamA POTRAs1-3 is to support a connection with the Sec translocon to form a transperiplasmic bridge, as well as provide a docking site for the chaperone SurA for substrate delivery.

  PublisherOA PDFDOI

• A Type III secretion system effector evolved to be mechanically labile and initiate unfolding from the N-terminus

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-12-04

  articleOpen accessSenior authorCorresponding

  Abstract Many Gram-negative pathogens critically depend on the Type III secretion system (T3SS) to inject effector proteins into host cells for colonization. Because the channel of the T3SS is narrow (∼2 nm), effectors must be unfolded for secretion. However, the T3SS cannot unfold mechanically robust substrates (GFP, ubiquitin, and dihydrofolate reductase), severely impairing their secretion. Consistent with this, effectors are exceptionally mechanically labile, unfolding at low forces. Thus, secretion competency is correlated with mechanical properties. Effector sequences have significantly diverged from non-effectors, suggesting that secretion exerts evolutionary pressure selecting mechanical lability. Here, using atomic-force-microscopy–based force spectroscopy, we show that effector NleC is mechanically labile ( F unfold = 13.5 pN at 100 nm/s) and mechanically compliant, as characterized by a large distance to the transition state (Δ x ‡ = 2.7 nm). In contrast, the non-effector homolog protealysin is mechanically stable ( F unfold = 50.7 pN at 100 nm/s) and brittle (Δ x ‡ = 0.7 nm), comparable to proteins known to impair secretion ( F unfold > 80 pN; Δ x ‡ < 0.4 nm). Denaturant-induced unfolding assays demonstrate that effectors exhibit rates typical of their fold, further reinforcing mechanical properties rather than fast unfolding kinetics ( k 0 ) predicts secretion. Steered molecular dynamic simulations revealed NleC unfolding initiates at the N-terminus, consistent with current secretion models, whereas protealysin unfolding initiates at the C-terminus. Notably, the NleC N-terminus is primarily α-helical while non-effector homologs contain β-sheets, which may account for the distinct unfolding pathway. Together, these results support the notion that mechanical lability is an evolved, structurally encoded feature underlying effector secretion. Significance The Type III secretion system (T3SS) delivers effector proteins directly into host cells to promote bacterial colonization. Effectors must be unfolded for secretion, and this particular selective pressure is hypothesized to have driven significant sequence divergence from non-effector proteins. Here, we show that effectors are not characterized by unusually fast unfolding rates. Rather as hypothesized, effector NleC is more mechanically labile than its non-effector homolog, indicating that mechanical lability underlies both effector sequence divergence and T3SS unfolding. Simulations revealed that NleC unfolding initiates via the N-terminus consistent with the current secretion mechanism, while protealysin unfolds from the C-terminus. Together, these results strongly suggest mechanical lability is an evolved property of effectors and provide structural insight into how it is encoded.

  PublisherDOI

• A fluorescent reporter and single-turnover kinetics reveal insight into BAM complex function

  Proceedings of the National Academy of Sciences · 2025-12-24

  articleOpen accessSenior authorCorresponding

  The β-barrel assembly machine (BAM) is an essential protein complex that folds and inserts outer membrane β-barrel proteins (OMPs) into the bacterial outer membrane. The BAM complex contains the essential BamA OMP with five soluble polypeptide transport-associated (POTRA) domains, which scaffold the essential BamD lipoprotein and the nonessential lipoproteins BamB/C/E. The importance of each BAM component has been investigated primarily using cell-based phenotypic assays, and structural data have revealed insights into the role of the BamA β-barrel in OMP folding. However, in vitro quantitative analysis for the function of each BAM component has been challenging. We describe the development of a fluorescent reporter of OMP folding, bOmpA-A488, which we use to obtain single-turnover kinetic parameters for wild-type BAM complex in vitro. We observe a k fold of 0.78 ± 0.15 min −1 and approximate substrate affinity of 3.1 ± 1.1 µM consistent with estimates of in vivo requirements. Furthermore, we find that while BamA alone is inactive in Escherichia coli lipid liposomes, BamAB and BamAD subcomplexes have activities similar to that of the holoBAM complex. This suggests that OMP folding and insertion is catalyzed by BamA while the accessory lipoproteins maintain BamA in a catalytically competent conformation. We also find that, contrary to prevailing models, POTRA domain deletions that include POTRA3, which is essential in cells, do not drastically impact activity. This indicates that the first three POTRA domains do not play a major role in binding or folding OMPs under single-turnover conditions, suggesting a different role in cells.

  PublisherDOI

• Synthesis of Nucleotide Diphosphate Uronic Acids via the Coupling of Activated Nucleotides with Uronic Acid-1-phosphates

  The Journal of Organic Chemistry · 2025-03-25

  article

  The stereoselective synthesis of nucleotide diphosphate (NDP) uronic acids from simple sugar precursors, including d-gluco-, d-galacto-, and d-mannopyranoside derivatives, is described. Key to this convergent synthesis is the coupling of unprotected uronic acid 1-phosphate with a nucleotide phosphorimidazolide to directly form the NDP-uronic acid, of which 11 derivatives were prepared. The coupling is compatible with the carboxylic acid functionality present in uronic acid-1-phosphates, with conversions of >95% and isolated yields typically above 60%. Key features of this work include (i) stereoselective synthesis of α-d-phosphoglycosides from perbenzylated α- and β-d-thioglycosides, (ii) selective and mild oxidation of galactose-, glucose-, and mannose-1-phosphates to the corresponding uronic acid-1-phosphate, and (iii) mild coupling conditions to directly provide nucleotide diphosphate uronic acids from unprotected uronic acid-1-phosphates and nucleotide phosphorimidazolides. This chemistry is currently in use to develop inhibitors of key enzymes involved in antibiotic resistance.

  PublisherDOI

• The Synthesis of Nucleotide Diphosphate Uronic Acids via the Coupling of Activated Nucleotides with Uronic Acid-1-Phosphates

  ChemRxiv · 2024-04-23 · 2 citations

  preprintOpen access

  The stereoselective synthesis of nucleotide diphosphate (NDP) uronic acids from simple sugar precursors, including gluco-, galacto-, and mannopyranoside derivatives, is described. Key to this convergent synthesis is the coupling of a uronic acid 1-phosphate with a nucleotide monophosphate activated as the phosphorimidazolide to form the NDP-uronic acid, of which 10 derivatives were prepared. The coupling is compatible with the carboxylic acid functionality present in uronic acid-1-phosphates with conversions above 95% and isolated yields typically above 60%. Key features of this work include (i) stereoselective synthesis of ⍺-phosphoglycosides from perbenzylated ⍺- and β-thioglycosides, (ii) selective and mild oxidation of galactose-, glucose-, and mannose-1-phosphates to the corresponding uronic acid-1-phosphate, and (iii) mild coupling conditions to directly provide nucleotide diphosphate uronic acids from unprotected uronic acid-1-phosphates and nucleotide phosphorimidazolides.

  PublisherOA PDFDOI

• Similarly slow diffusion of BAM and SecYEG complexes in live E. coli cells observed with 3D spt-PALM

  Biophysical Journal · 2023-10-17 · 5 citations

  articleOpen accessSenior authorCorresponding
  PublisherOA PDFDOI

• Structure and Function of ArnD. A Deformylase Essential for Lipid A Modification with 4-Amino-4-deoxy-<scp>l</scp>-arabinose and Polymyxin Resistance

  Biochemistry · 2023 · 11 citations

  Senior authorCorresponding
  • Biochemistry
  • Biology
  • Chemistry

  , purified stArnD efficiently deformylates C55P-Ara4FN confirming its role in Ara4N biosynthesis. Mutations D9N and H233Y completely inactivate stArnD implicating these two residues in a metal-assisted acid-base catalytic mechanism.

  PublisherOA PDFDOI

• Targeting the Conformational Change in ArnA Dehydrogenase for Selective Inhibition of Polymyxin Resistance

  Biochemistry · 2023 · 5 citations

  Senior authorCorresponding
  • Chemistry
  • Biochemistry
  • Stereochemistry

  binding and catalysis. Enzyme activity and binding assays show that (i) UDP-GlcA analogs lacking the 6' carboxylic acid bind the enzyme but fail to trigger the conformational change, resulting in poor inhibition, and (ii) the uridine monophosphate moiety of the substrate provides most of the ligand binding energy. Mutation of asparagine 492 to alanine (N492A) disrupts the ability of ArnA_DH to undergo the conformational change while retaining substrate binding, suggesting that N492 is involved in sensing the 6' carboxylate in the substrate. These results identify the UDP-GlcA-induced conformational change in ArnA_DH as an essential mechanistic step in bacterial enzymes, providing a platform for selective inhibition.

  PublisherOA PDFDOI

• Type III secretion system effector proteins are mechanically labile

  Proceedings of the National Academy of Sciences · 2021 · 54 citations

  Senior authorCorresponding
  • Cell biology
  • Biology
  • Chemistry

  < 0.4 nm). These results suggest that effector protein unfolding by T3SS is a mechanical process and that mechanical lability facilitates efficient effector protein secretion.

  PublisherDOI

• Biochemical and Structural Characterization of Enzymes Responsible of Polymyxin Resistance in Gram-Negative Bacteria

  Biophysical Journal · 2020-02-01 · 1 citations

  articleOpen accessSenior author
  PublisherOA PDFDOI

Recent grants

• NIH Grant R01EY019050

  NIH · $874k · 2013

• NIH Grant R01AI060841

  NIH · $1.4M · 2009

• NIH Grant R56AI060841

  NIH · $747k · 2012

• NIH Grant R01AI080709

  NIH · $2.6M · 2018

• NIH Grant P41RR001209

  NIH · $12.9M · 2015

Frequent coauthors

• Thomas T. Perkins

  National Institute of Standards and Technology

  12 shared

• Devin T. Edwards

  National Institute of Standards and Technology

  8 shared

• Petia Z. Gatzeva-Topalova

  University of Colorado Boulder

  8 shared

• Marc-André LeBlanc

  8 shared

• Robert Walder

  7 shared

• Pamela A. Doerner

  University of Colorado Boulder

  7 shared

• Krzysztof Palczewski

  University of California, Irvine

  6 shared

• Austin Bargmann

  University of Colorado System

  5 shared