If your system is busy, the top level directory is likely to end up in buffer cache, no matter where it is rooted. (If it's not busy, a few extra milliseconds won't matter). Take a look at directory sizes for 1-digit/2-digit/3-digit prefixes. A "sweet spot" would be directories that (just) fit into whatever block size your filesystem uses, often 4KB. I'm guessing that will be 2- or 3-digit prefixes, depending on how dense the prefixes are and how the file system structures directories. You might let the top level directory get a bit larger, on the grounds that frequent access will keep it pinned in buffer cache.
Don't go nuts with premature optimization. Make it easy to alter the structure of the directories, like having a routine to return an array of components corresponding to the directory entries. Then measure performance with a few alternatives.