Topics (Young Scientist Series)
Functional and biophysical characterization of abundant transport proteins from SAR11 bacteria
2025 Volume 65 Issue 3 Pages 156-158
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2025 Volume 65 Issue 3 Pages 156-158
海洋細菌の輸送タンパク質は，海洋における栄養素循環に重要である．本稿では，遍在するSAR11細菌の輸送タンパク質の機能解析について紹介する．非常に高い結合親和性に代表される，これらの輸送タンパク質に特有な生物物理学的特性が，SAR11細菌の低栄養環境への適応に大きく関与していることが明らかになった．
Marine microorganisms constitute the majority of ocean biomass and have a global impact on the Earth’s ecosystems, geochemistry, and climate. Our knowledge of the diversity, biochemical activity, and biogeochemical impact of marine microbial communities has been dramatically advanced by a wealth of ocean metagenome and metatranscriptome data (i.e., next-generation sequencing data of DNA and RNA extracted from environmental samples) collected over the past two decades. However, accurate interpretation of gene distribution and expression patterns in metagenome and metatranscriptome datasets requires knowledge of gene function. This is a major challenge, because the discovery of novel genes far outpaces our ability to experimentally characterize their functions. Even well-characterized protein families can show enormous amounts of hidden functional diversity, limiting the accuracy of homology-based predictions of gene function. These qualitative predictions also provide no information on important biochemical and biophysical properties of proteins that may be relevant for microbial adaptation to different environments or ecological niches, such as kinetic parameters and allosteric regulation for enzymes, uptake rate and affinity for transporters, and spectroscopic properties for light-harvesting proteins. Thus, experimental characterization of protein function is essential for extracting the maximum value from existing ocean metagenome and metatranscriptome data. But where to start?
Transport proteins are valuable and particularly tractable targets for large-scale functional characterization1). Transport proteins are positioned at the interface of marine microbes with their environment; they mediate uptake of dissolved organic matter (DOM) and thereby contribute to fluxes of carbon, sulfur, nitrogen, and phosphorus in the environment. They also mediate metabolic exchange and communication in microbial communities. Transport genes are often co-localized with genes required for catabolism of the transported substrate; thus, functional annotation of transport proteins can also guide discovery of metabolic pathways based on genome context1). Altogether, transport proteins can provide insight into the physiology, ecology, metabolism, and chemical environment of marine microbes. In our recent work2), we focused on characterization of solute-binding protein (SBP)-dependent transporters in Candidatus Pelagibacter ubique HTCC1062, a representative of the ubiquitous SAR11 clade of marine bacteria.
SAR11 bacteria are chemoheterotrophic bacteria that are highly abundant throughout the surface ocean. They are oligotrophic (i.e., adapted to low-nutrient conditions) and specialize in harvesting simple metabolites that are widely available in the ocean albeit at low concentrations3). One factor in the success of SAR11 bacteria in this ecological niche appears to be its reliance on SBP-dependent transporters. SBPs are soluble extracytoplasmic proteins that bind specific substrates with high affinity and mediate their uptake across the inner membrane together with various families of membrane transport proteins (Fig. 1). SBPs are abundant in the SAR11 proteome4) and are accommodated in an unusually large periplasm (up to 70% of cell volume5)). Given the high abundance of SBPs in SAR11 bacteria and the abundance and nutrient uptake activity of SAR11 bacteria in the surface ocean, the SBP-dependent transporters of SAR11 bacteria likely contribute significantly to uptake of key components of low-molecular-weight (LMW) DOM in the surface ocean, resulting in broader biogeochemical significance. However, due to the difficulty of SAR11 cultivation and a lack of genetic manipulation tools, our knowledge of the functions and properties of these transport proteins was limited, with the notable exception of a high-affinity multifunctional osmolyte transporter that had been characterized at the cellular level6).
Structural architecture of the three main SBP-dependent transporter families: ATP-binding cassette (ABC), tripartite ATP-independent transporter (TRAP), and tripartite tricarboxylate transporter (TTT) families. TRAP and TTT transporters have similar architectures. Created with Biorender.com.
In our recent work2), we therefore aimed to perform functional characterization of all eighteen SBPs in a representative SAR11 bacterium, Ca. P. ubique HTCC1062. Thirteen SBPs could be assigned a function based on high-throughput screening against metabolite libraries using differential scanning fluorimetry, followed by validation with isothermal titration calorimetry (ITC), X-ray crystallography, and/or gas chromatography-mass spectrometry. Notably, we were able to assign functions to some SBPs that are highly abundant in the ocean metatranscriptome, such as the osmolyte-binding protein SAR11_1336, which we estimated to account for roughly 5% of SBP transcripts in the surface ocean2).
From a biophysical perspective, a striking finding was that the SBPs from SAR11 bacteria have unusually high binding affinity. SBPs usually bind their highest affinity ligand with a Kd between 10 nM and 1 μM. A systematic survey of literature data (n = 206) showed a median Kd value of 165 nM and a lower limit of ~200-400 pM2). In contrast, five of the SBPs from SAR11 bacteria exhibited picomolar Kd values; values as low as 20-30 pM were observed for multiple SBPs. These Kd values are too low to be measured by direct ITC experiments and were therefore validated through competitive ITC experiments. For example, SAR11_1210 was shown to have a Kd value of 32 pM for l-arginine through protein-protein competition experiments using a previously characterized l-arginine-binding protein (Fig. 2a). Altogether, these results showed that adaptations in the biophysical properties of SBPs act together with adaptations in cellular characteristics (small size, large periplasm, high SBP expression) to make SAR11 bacteria highly successful competitors for valuable but scarce components of LMW DOM.
(a) Competitive binding of l-arginine to SAR11_1210 and a homologous l-arginine-binding protein, SeLAOBP. This ITC experiment provides direct evidence that the Kd of SAR11_1210 is several orders of magnitude lower than the Kd of SeLAOBP (15 nM). (b) SBPs undergo a conformational change upon binding. The free energy change of binding can be decomposed into separate contributions from the conformational change (ΔGoclosing) and the protein-ligand interactions (ΔGcbinding). (c) Structural comparison of SAR11_1210 (left) with a homologous l-arginine binding protein (right; PDB ID: 2Q2A, Kd 39 nM7)). Figure adapted from 2).
To investigate the structural basis for high binding affinity, we solved a high-resolution (1.32 Å) crystal structure of SAR11_1210 complexed with l-arginine. The binding mode of l-arginine to SAR11_1210 was similar to homologous l-arginine-binding proteins (Fig. 2c). However, the structure of SAR11_1210 showed an unusual feature: a direct interaction between the ligand and the hinge region of the protein, mediated by Glu108. The hinge region of SBPs mediates a large-scale conformational change between an open conformation in the apo state and a closed conformation in the holo state (Fig. 2b). Mutations in the hinge region of SBPs have been shown to control binding affinity by adjusting the position of the intrinsic conformational equilibrium8). We hypothesize that the interaction between the ligand and Glu108 in SAR11_1210 stabilizes the hinge region and contributes to the stability of the closed conformation in the presence of ligand, thereby increasing binding affinity through a conformational coupling mechanism. Consistent with this hypothesis, the substitution Glu108Ala resulted in a 210-fold decrease in binding affinity. However, further computational and experimental exploration of conformational dynamics, combined with mutagenesis and evolutionary analyses, will be needed to fully rationalize the high binding affinity of SAR11_1210 and other SAR11 SBPs.
Our results showed how adaptation of biophysical properties such as binding affinity and specificity in SBPs contributed to the evolutionary success of SAR11 bacteria in their oligotrophic niche. These results also raise various questions for future research on structure and function of SBP-dependent transporters, for example: (1) Does evolution control binding affinity through modulation of the conformational landscape? (2) Is there a negative tradeoff between binding affinity and other relevant properties such as binding kinetics, uptake rate, or uptake efficiency? (3) Do adaptations in the transmembrane components of SBP-dependent transporters also affect uptake affinity? (4) How does evolution increase binding affinity while maintaining broad binding specificity? Thus, the unique properties of the SAR11 SBPs provide an opportunity to address general questions about the mechanisms of SBP-dependent transporters and the mechanisms that regulate binding affinity in protein evolution.
I thank Prof. Paola Laurino for her supervision of the work described in this article. The work described was funded by the Okinawa Institute of Science and Technology and supported by a JSPS Postdoctoral Fellowship for Overseas Researchers.
Ben E. CLIFTON
Lecturer, School of Molecular Sciences, University of Western Australia
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