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A dichotomous family of Cache domain proteins evolved through c-type heme acquisition

Maithili DeshpandeiD, Jiawei XingiD, Vadim M. GumeroviD, Robert DunleavyiD, Herbert Castillo, Brian R. CraneiD, Igor B. ZhuliniD

PDFDOI

In-depth summary

Claude Opus 5.5 · xhigh effort · 7 Nov 2026 · from the full text of version 2 (1 Oct 2026) · CC BY 4.0

Cache sensor domains of DUF3365/Tll0287 family bind covalent c-type heme through a CXXCH insertion; a signaling member behaves like an NO sensor, a stand-alone one like a redox protein.

Cache domains were thought to act only as ligand-binding modules, and the DUF3365/Tll0287 family had been annotated as PAS-like. The paper reassigns the family to Cache and proposes that a short cytochrome c-derived fragment was inserted into a pre-existing Cache scaffold.

Key results

  1. ShownThe CXXCH c-type heme-binding motif is almost invariant across the DUF3365/Tll0287 family, so covalent heme binding defines the family.

    Motif conserved in 99% of Dataset 1 (more than 5,000 InterPro sequences) and 99.4% of Dataset 2 (about 20,000 PSI-BLAST hits against Clustered NR). · The DUF3365/Tll0287 domain family contains a…

  2. ShownThe family belongs to the Cache superfamily rather than PAS, and differs from canonical single-module Cache domains by a heme-bearing insertion; the authors name it sCache_heme.

    All ten top DALI matches to the Tll0287 structure (PDB 5b82) were annotated as Cache; HHpred also gave Cache profiles. The insertion lies between strands β3 and β4 and carries the CXXCH motif. · DUF3365/Tll0287 is a cofactor-containing Cache…

  3. ShownsCache_heme is enriched in Gracilicutes, and nearly half of the domains are stand-alone, unlike most Cache domains. The family splits into Cluster 1 (stand-alone or cytochrome c-fused) and Cluster 2 (signaling fusions).

    More than 50% of genomes in several phyla (e.g. Nitrospirota, Desulfobacterota) encode one; more than 90% of Cache domains are usually fused to signaling modules. Cluster 1 and 2 differ at specific conserved positions (e.g. position 44 hydrophobic in 97% of Cluster 1). · Phyletic distribution and domain architecture of…; Residue conservation within the two clusters of…

  4. SuggestedSequence searches support a model in which a cytochrome c-like fragment with CXXCH was inserted into an existing stand-alone sCache domain; stand-alone forms are proposed as ancestral.

    A 13-residue GSU0582 segment hit cytochromes in BLAST. PSI-BLAST with that segment deleted retrieved a DcuS/MalK-type kinase sCache domain lacking CXXCH (E value 3.28 × 10⁻¹⁷, sixth iteration). · Origin of the heme c-binding motif in Cache…; DISCUSSION

  5. SuggestedTwo recombinant representatives differ in NO and redox behaviour: the signaling-associated P. azotoformans protein binds NO tightly, while the stand-alone P. putida protein binds it weakly and has a cytochrome-like reduction potential.

    NO koff (6.25 ± 0.64) × 10⁻⁴ s⁻¹ for P. azotoformans versus rapid dissociation for P. putida. Reduction potential 123 mV (WT) and 113 mV (C59A) for P. putida. Reduced-state autoxidation (4.78 ± 0.83) × 10⁻² s⁻¹ versus (0.5 ± 0.1) × 10⁻² s⁻¹. · Spectroscopic characterization of redox states; NO binding and oxygen reactivity distinguishes…

Methods, data and reproducibility

Sequence, profile and structure comparisons (InterPro, PSI-BLAST, DALI, HHpred, GTDB taxonomy) defined the family, its distribution and its likely origin. One protein from each architectural cluster was produced in E. coli and characterised by UV-visible spectroscopy, NO off-rate, autoxidation and reduction-potential measurements.

  • Dataset 1: Tll0287-like domain sequences curated from InterPro · bacteria · n = more than 5,000 sequences
  • Dataset 2: PSI-BLAST hits against NCBI Clustered NR using Tll0287 as query · bacteria (a few archaeal homologs) · n = about 20,000 sequences
  • Recombinant stand-alone sCache_heme protein (Target 1) from Pseudomonas putida · Pseudomonas putida · n = one protein plus a C59A variant · WP_080580316
  • Recombinant full-length membrane sCache_heme protein (Target 2) from Pseudomonas azotoformans · Pseudomonas azotoformans · n = one protein · WP_033902098
  • SEC-SAXS data for the P. azotoformans protein (stable dimer in detergent) · Pseudomonas azotoformans · SASD2G3
Methods & tools
MiST 4.0, InterPro/Pfam, BLAST/PSI-BLAST, DALI, HHpred, GTDB release 10, iTOL v.6, MAFFT v7.490, Jalview, WebLogo 3, AlphaFold 3, BioXTAS RAW
Reagents & environment
MAFFT v. 7.490, Jalview v. 2.11.3.064, iTOL v.6, GTDB release 10, WebLogo 3.
Reproducibility gaps
No code or analysis scripts are mentioned. Datasets S1-S3 and the supplementary tables are said to be in the Supplementary Information; only the SAXS data have a named public accession.
Limitations (4)
  • Redox and oxygen-binding parameters of recombinantly expressed, purified heme proteins can be misleading for assigning sensing functions. · stated by the authors
  • The true functions of the stand-alone sCache_heme proteins remain unclear, and the functional significance of the cluster-specific conserved positions is unknown. · stated by the authors
  • Only two proteins (one per cluster), both produced in E. coli, were characterised, so the functional split between clusters rests on very few examples. · noted in this summary
  • The cytochrome-origin model and the stand-alone ancestral state are inferred from sequence-similarity searches and parsimony, not from an explicit evolutionary reconstruction. · noted in this summary

Read this if you study bacterial sensor domains, heme-based sensing or Cache/PAS domain evolution and want a classification of the DUF3365/Tll0287 family.

Citations
1

Abstract

PAS domains often bind cofactors, whereas related Cache domains are thought to function exclusively as ligand-binding modules. We identify and characterize a large family of Cache domains containing covalently bound c-type heme. Sequence, phylogenetic and structual analysis of the DUF3365/Tll0287 family reveals conservation of a canonical CXXCH heme-binding motif, establishing c-type heme binding as a defining feature of this family. Despite previous annotation as PAS-like, these proteins share a distinct heme-binding insertion into a pre-existing Cache scaffold, representing the first documented recruitment of an intact cofactor-binding motif into a bacterial sensor domain. Domain architecture further divides the family into two subclasses: one exhibiting fusions to signaling domains and another comprised of standalone proteins. Spectroscopic analyses indicate that a representative signaling protein acts as a nitric oxide sensor, whereas a stand-alone representative exhibits redox-related properties and weak nitric oxide binding. These findings expand the functional repertoire of Cache domains and reveal a mechanism for the evolution of bacterial sensory systems.

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