We have developed a new in-cell NMR method that is applicable to any type of cell and does not require target protein modification or specialized equipment. The stable-isotope-labeled target protein, thymosin beta4 (Tbeta4), was delivered to 293F cells, which were permeabilized by a pore-forming toxin, streptolysin O, and resealed by Ca(2+) after Tbeta4 uptake. As a result, we successfully observed (1)H-(15)N HSQC signals originating from the Tbeta4, including those from the N-terminal acetylation, which had occurred inside the cell as a post-translational modification.

CD44, a major cell surface receptor for hyaluronan (HA), contains a functional domain responsible for HA binding at its N terminus (residues 21-178). Accumulating evidence indicates that proteolytic cleavage of CD44 in its extracellular region (residues 21-268) leads to enhanced tumor cell migration and invasion. Hence, understanding the mechanisms underlying the CD44 proteolytic cleavage is important for understanding the mechanism of CD44-mediated tumor progression. Here we present the NMR structure of the HA-binding domain of CD44 in its HA-bound state. The structure is composed of the Link module (residues 32-124) and an extended lobe (residues 21-31 and 125-152). Interestingly, a comparison of its unbound and HA-bound structures revealed that rearrangement of the ␤-strands in the extended lobe (residues 143-148) and disorder of the structure in the following C-terminal region (residues 153-169) occurred upon HA binding, which is consistent with the results of trypsin proteolysis studies of the CD44 HA-binding domain. The order-to-disorder transition of the C-terminal region by HA binding may be involved in the CD44-mediated cell migration.

The hyaluronan (HA) receptor CD44 mediates cell adhesion in leukocyte trafficking and tumor metastasis. Our previous nuclear magnetic resonance (NMR) studies revealed that the CD44 hyaluronan-binding domain (HABD) alters its conformation upon HA binding, from the ordered (O) to the partially disordered (PD) conformation. Here, we demonstrate that the HABD undergoes an equilibrium between the O and PD conformations, in either the presence or absence of HA, which explains the seemingly contradictory X-ray and NMR structures of the HA-bound HABD. An HABD mutant that exclusively adopts the PD conformation displayed a higher HA affinity than the wild-type. Rolling of the cells expressing the mutant CD44 was less efficient than those expressing the wild-type, due to the decreased tether frequency and the slow cellular off rate. Considering that the mutant CD44, devoid of the low-affinity state, exhibited impaired rolling, we conclude that the coexistence of the high- and low-affinity states of the HABD is essential for the CD44-mediated rolling.

The authors note that Fig. 4 and Fig. S4 appeared incorrectly. The corrected figures and their legends appear below. The SI has been corrected online.The authors also note that on page 1 of the supporting information, right column, second full paragraph, lines 5-13, "The starting structure was solvated in a 62 × 129 × 69-Å TIP3 water box (6) with 54 chloride ions to neutralize the system, resulting in 53,872 atoms. The system was minimized for three consecutive 500-conjugate gradient steps, during which the protein was first held fixed; next, only the heavy atoms of the protein were held fixed, and finally, all atoms were allowed to move. After the energy minimizations, the system was heated gradually from 0 K to 310 K over 52 ps, and the temperature was maintained using Langevin dynamics" should instead appear as "The starting structure was solvated in a 68 × 116 × 79-Å TIP3 water box (6) with 53 chloride ions to neutralize the system, resulting in 59,606 atoms. The system was minimized for two consecutive 500-conjugate gradient steps, during which the protein was first held fixed; next, only the heavy atoms of the protein were held fixed. After the energy minimizations, the system was heated gradually from 0 K to 310 K over 9.3 ps, and the temperature was maintained using Langevin dynamics. Subsequently, the system was equilibrated by 4 rounds, first the heavy atoms were held fixed, then only backbone atoms, then only Cα atoms, and finally all atoms were allowed to move." These errors do not affect the conclusions of the article.A B C Fig. 4. Steered molecular dynamics (SMD) simulation of CD44 HABD by pulling force. (A) Snapshots of the HABD-HA8 complex at 0 ns (Left) and 3.5 ns (Right) during the SMD simulation. In the SMD simulation, the Cα carbon of the C-terminal residue Ile173 (gray sphere) was pulled at 10 Å/ns in the indicated direction (arrow), whereas the C2 atom of the N-acetylglucosamine residue (green sphere) was kept fixed. In the snapshot after 3.5 ns in the SMD simulation, the C-terminal mechanosensitive latch was completely separated from the α1 helix. (B) The time course of the hydrogen bond donor-acceptor distances between HA NAG1177 and I100 (red) and E52 and Y166 (blue). (C) Schematic depiction of the mechanosensitive latch in the force-induced conformational change of CD44 HABD.

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