The green fluorescent protein (GFP)-nanobody is a single-chain V H H antibody domain developed with specific binding activity against GFP and is emerging as a powerful tool for isolation and cellular engineering of fluorescent protein fusions in many different fields of biological research. Using X-ray crystallography and isothermal titration calorimetry, we determine the molecular details of GFP:GFP-nanobody complex formation and explain the basis of high affinity and at the same time high specificity of protein binding. Although the GFP-nanobody can also bind YFP, it cannot bind the closely related CFP or other fluorescent proteins from the mFruit series. CFP differs from GFP only within the central chromophore and at one surface amino acid position, which lies in the binding interface. Using this information, we have engineered a CFP variant (I146N) that is also able to bind the GFP-nanobody with high affinity, thus extending the toolbox of genetically encoded fluorescent probes that can be isolated using the GFP-nanobody.

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Summary Plasminogen activator inhibitor-1 (PAI-1) has a pro-tumorigenic function via its pro-angiogenic and anti-apoptotic activities. Here we demonstrate that PAI-1 promoted the recruitment and M2 polarization of monocytes/macrophages through different structural domains. Its LRP1 interacting domain regulated macrophage migration, while its C-terminal uPA interacting domain promoted M2 macrophage polarization through activation of p38MAPK and NF-κB and induction of an autocrine IL-6/STAT3 activation pathway. We then show in several experiments in mice, that expression of PAI-1 is associated with increased tumorigenicity, increased presence of M2 macrophages, higher levels of IL-6 and increased STAT3 phosphorylation in macrophages. Strong positive correlations between PAI-1, IL-6 and CD163 (M2 marker) expression were also found by meta-analysis of transcriptome data in many human cancers. Altogether, these data provide evidence for a mechanism explaining the paradoxical pro-tumorigenic function of PAI-1 in cancer.